Electrochemical diagnostic system

ABSTRACT

Described are devices, systems, kits, methods, and techniques for analyzing test fluids to determine presence, absence, or concentration of analytes in the test fluids and useful for performing diagnostic testing, such as to quickly identify whether or not an individual may have a particular disease or condition, such as infection by SARS-CoV-2 or a SARS-CoV-2 variant or vaccine-induced immunity or natural immunity to infection by SARS-CoV-2 or a SARS-CoV-2 variant. The devices, systems, kits, methods, and techniques described herein can be used to detect analytes in body fluids using a functionalized electrochemical test strip and potentiostatic measurements, allowing for prompt identification of whether or not an individual has a particular disease or condition, such as in a period of 5 minutes or less.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. ProvisionalApplication No. 63/201,982, filed on May 21, 2021, U.S. ProvisionalApplication No. 63/202,135, filed on May 28, 2021, U.S. ProvisionalApplication No. 63/202,439, filed on Jun. 11, 2021, U.S. ProvisionalApplication No. 63/202,828, filed on Jun. 25, 2021, U.S. ProvisionalApplication No. 63/203,009, filed on Jul. 3, 2021, U.S. ProvisionalApplication No. 63/232,720, filed on Aug. 13, 2021, and U.S. ProvisionalApplication No. 63/235,433, filed on Aug. 20, 2021, which are herebyincorporated by reference in their entireties.

TECHNICAL FIELD

This invention is in the field of diagnostic testing. This inventionrelates generally to electrochemical diagnostic test systems andcomponents and techniques for rapidly identifying the presence ofdiagnostic analytes in body fluid samples.

BACKGROUND

Present and future containment of pathogen outbreaks such as COVID-19are dependent on testing and retesting on very wide-scales. In a globalpandemic this translates to a need for billions of rapid, accurate, andinexpensive tests performed at millions of locations. Further, datacollection and aggregation should be nearly real-time to meet the needsof crisis managers. Current polymerase chain reaction (PCR) testingmethods used for genomic analysis generally utilize sample collectionfollowed by transport prior to PCR amplification and sequencing, whichcan delay determination of results. Rapid testing using antigen testingtends to be less specific, but can provide qualitative positive negativeresults in a much quicker time period (e.g., within 15 minutes).Improvements to viral testing methods are needed.

Both previous infection and vaccination have been shown to provideprotection from COVID-19. However, waning immunity and viral variationmay lead to a loss of protection over time. There are several predictivemodels of immune protection in terms of antibody levels over timedeveloped in an attempt to identify immune correlates of protection toassist in determining susceptibility to infection post vaccination andthe need to administer vaccine booster doses. Current measurement ofanti-COVID-19 antibody levels follow a serology approach and test bloodsamples. These tests detect antibodies (for example, IgM or IgG) to theSARS-CoV-2 virus and require a blood draw at a point of care visit,which is sent to a lab and results take up to 3 days to receive.However, serum antibody levels are not routinely ordered by physiciansto monitor protection or when there is an exposure concern.

Overall, there is no device that can rapidly identify the immunecorrelate(s) of protection from SARS-CoV-2 infection or any method forpredicting how changes in antibody levels will be reflected in clinicaloutcomes. Further, there is no simple, rapid method available to assessantibody levels post vaccination or to differentiate between immuneresponses after vaccination vs. the responses in convalescentindividuals due to prior infection.

SUMMARY

In a first aspect, electrochemical test strip devices are described. Anexample electrochemical test strip device of this aspect comprises afirst set of electrodes including a first reference electrode, a firstworking electrode, and a first counter electrode, the first workingelectrode functionalized with active capture molecules, such as activecapture molecules that include one or more electroactive redox tagsand/or active capture that molecules bind or are configured to bind atarget analyte; a second set of electrodes including a second referenceelectrode, a second working electrode, and a second counter electrode,the second working electrode functionalized with null capture molecules,such as null capture molecules that include one or more electroactiveredox tags and/or null capture molecules that bind or are configured tobind different analytes than the active capture molecules or do not bindor are not configured to bind the target analyte; a fluid chamber influid communication with the first set of electrodes and the second setof electrodes, the fluid chamber for receiving a test fluid with orwithout a test analyte and establishing contact between the test fluidand the first set of electrodes and between the test fluid and thesecond set of electrodes; a first set of electrode contacts inelectrical communication with the first set of electrodes, the first setof electrode contacts including a first reference electrode contact, afirst working electrode contact, and a first counter electrode contact;and a second set of electrode contacts in electrical communication withthe second set of electrodes, the second set of electrode contactsincluding a second reference electrode contact, a second workingelectrode contact, and a second counter electrode contact. In examples,the first working electrode includes a first mixedself-assembled-monolayer (SAM) including a first linker component and acharged passivation component, the first linker component terminallybinding the active capture molecules. In examples, the second workingelectrode includes a second mixed SAM including a second linkercomponent and the charged passivation component, the second linkercomponent terminally binding the null capture molecules. In examples,the first mixed SAM and the second mixed SAM are coated with or comprisean anti-fouling hydrogel comprising a plurality of different chargedglycosaminoglycans.

The electrochemical test strip devices described herein are useful for avariety of applications. For example, the electrochemical test stripdevice is or comprises an antigen assay for a virus or a componentthereof. Optionally, the electrochemical test strip device is orcomprises an antigen assay for a viral biomarker. Optionally, theelectrochemical test strip device is or comprises an antigen assay foran inflammatory biomarker. In examples, the target analyte is the virusor the component thereof, the viral biomarker, or the inflammatorybiomarker. Optionally, the active capture molecules include a firstantibody that binds the virus or the component thereof, the viralbiomarker, or the inflammatory biomarker. Optionally, the active capturemolecules include a receptor protein that binds the virus or thecomponent thereof, the viral biomarker, the inflammatory biomarker. Inexamples, the null capture molecules include a second antibody that doesnot bind the virus or the component thereof, the viral biomarker or theinflammatory biomarker.

The electrochemical test strip devices of this aspect are useful forrapidly detecting infection by a virus. For example, the virus may be acoronavirus, the viral biomarker may be a coronavirus spike protein or acoronavirus nucleocapsid protein, or the inflammatory biomarker may be acoronavirus-induced inflammatory biomarker or host protein. Inparticular examples, the virus is SARS-CoV-2 or a variant of SARS-CoV-2,the viral biomarker comprises a spike protein or a nucleocapsid proteinof SARS-CoV-2 or a variant of SARS-CoV-2, or the inflammatory biomarkercomprises a cytokine, a SARS-CoV-2-mediated inflammatory biomarker, or aSARS-CoV-2 variant-mediated inflammatory biomarker. In some examples,the first antibody comprises an anti-SARS-CoV-2 antibody, ananti-SARS-CoV-2 variant antibody, or an anti-cytokine antibody.Optionally, when a receptor protein is used, the receptor protein maycomprise an angiotensin-converting enzyme 2 (ACE-2) or portion thereof.Such configurations may be useful, for example, for identifyinginfection by SARS-CoV-2 or a variant of SARS-CoV-2.

In another example, the electrochemical test strip device is orcomprises an assay for detection of viral antibodies or antiviralvaccine-induced antibodies. For example, the target analyte may be aviral infection-developed antibody or an antiviral vaccine-developedantibody, and the active capture molecules include a viral structuralprotein or portion thereof. In some cases, the viral infection-developedantibody comprises a coronavirus infection-developed antibody, whereinantiviral vaccine-developed antibody comprises an anti-coronavirusvaccine-developed antibody, or the viral structural protein comprises acoronavirus structural protein.

In some specific examples, the viral infection-developed antibodycomprises a SARS-CoV-2 infection-developed antibody or a SARS-CoV-2variant infection-developed antibody, the antiviral vaccine-developedantibody comprises an anti-SARS-CoV-2 vaccine-developed antibody oranti-SARS-CoV-2 variant vaccine-developed antibody, or the viralstructural protein comprises a SARS-CoV-2 or SARS-CoV-2 variant spikeprotein, a SARS-CoV-2 or SARS-CoV-2 variant nucleocapsid protein, or aSARS-CoV-2 or SARS-CoV-2 variant envelope protein. Optionally, the viralstructural protein comprises a SARS-CoV-2 spike protein, a SARS-CoV-2nucleocapsid protein, or a SARS-CoV-2 envelope protein. Optionally, thenull capture molecules include a SARS-CoV-2 variant spike protein orportion thereof, a SARS-CoV-2 variant nucleocapsid protein or portionthereof, or a SARS-CoV-2 variant envelope protein or portion thereof.Such configurations may be useful, for example, for identifying priorinfection by SARS-CoV-2 or a variant of SARS-CoV-2 or vaccinationagainst SARS-CoV-2 or a variant of SARS-CoV-2.

The electrochemical test strip devices described herein are not sospecific for use in detection of viruses and can be broadly applied to avariety of different systems. In some examples, the active capturemolecules include a first antibody, first receptor protein, or a firstaptamer that binds the target analyte and the null capture moleculesinclude a second antibody, second receptor protein, or a second aptamerthat does not bind the target analyte.

Without wishing to be bound by any theory, binding of the target analyteto the active capture molecules can modulates a proximity between thefirst working electrode and the one or more electroactive redox tags ofthe active capture molecules, such as relative to a proximity betweenthe second working electrode and the one or more electroactive redoxtags of the null capture molecules. An increase in hydrodynamic drag dueto the binding of the target analyte may impact the ability of theproximity between the first working electrode and the one or moreelectroactive redox tags of the active capture molecules to be modulatedby application of a potential to the first working electrode. In someexamples, the modulation that occurs upon binding the target analyte tothe active capture molecules can be observed as a modulation in currentmeasured in a voltammogram using the first set of electrodes, such as ina potentiostatic measurement system.

Preparation of the working electrodes of the electrochemical test stripdevices described herein can include preparing mixed SAMs that areadapted to couple to the active capture molecules. For example, one orboth the first linker component or the second linker componentoptionally comprises surface-bound alkyne terminated polyethylene glycol(PEG) molecules. Such alkyne functionalization may allow for reactionwith azido functionalized capture molecules, using click chemistry. Forexample, the first linker component and active capture molecule maycomprise an adduct or bioconjugate of an alkyne terminated polyethyleneglycol (PEG) molecule and an azido modified active capture molecule, theazido modified active capture molecule comprising an azido moiety, theone or more electroactive redox tags, and one or more binding regionsfor the target analyte. Optionally, the alkyne terminated polyethyleneglycol (PEG) molecule is immobilized on the surface of the first workingelectrode, and the alkyne terminated PEG molecule is coupled to theazido modified active capture molecule by a click chemistry reaction. Insome examples, the azido modified active capture molecule comprises anantibody including the azido moiety at a glycosylated region on acrystallizable fragment (Fc) portion of the antibody and the one or morebinding regions at an antigen-binding fragment (Fab) portion of theantibody. In another example, the second linker component and nullcapture molecule may comprise an adduct or bioconjugate of an alkyneterminated polyethylene glycol (PEG) molecule and an azido modified nullcapture molecule, the azido modified null capture molecule comprising anazido moiety, the one or more electroactive redox tags, and a bindingmoiety that does not bind the target analyte. In some examples, thealkyne terminated polyethylene glycol (PEG) molecule is immobilized onthe surface of the second working electrode, and wherein the alkyneterminated PEG molecule is coupled to the azido modified null capturemolecule by a click chemistry reaction.

The working electrode may include components that can limit, minimize,or avoid fouling, such as due to the presence of non-specific proteinsor substances in the test fluid. For example, the charged passivationcomponent optionally comprises charged polyethylene glycol (PEG)molecules bound to a surface of the first working electrode, such as PEGmolecules comprising one or more carboxylic acid groups or carboxylategroups. Optionally, the first mixed SAM comprises from about 10% toabout 50% of the first linker component, such as from 10% to 15%, from15% to 20%, from 20% to 25%, from 25% to 30%, from 30% to 35%, from 35%to 40%, from 40% to 45%, or from 45% to 50%. Optionally, the first mixedSAM comprises from about 50% to about 90% of the charged passivationcomponent, such as from 50% to 55%, from 55% to 60%, from 60% to 65%,from 65% to 70%, from 70% to 75%, from 75% to 80%, from 80% to 85%, orfrom 85% to 90%. Optionally, the second mixed SAM comprises from about10% to about 50% of the second linker component, such as from 10% to15%, from 15% to 20%, from 20% to 25%, from 25% to 30%, from 30% to 35%,from 35% to 40%, from 40% to 45%, or from 45% to 50%. Optionally, thesecond mixed SAM comprises from about 50% to about 90% of the chargedpassivation component, such as from 50% to 55%, from 55% to 60%, from60% to 65%, from 65% to 70%, from 70% to 75%, from 75% to 80%, from 80%to 85%, or from 85% to 90%. In some examples, a percent of a mixed SAMrefers to a mole fraction or an area fraction.

A variety of different charged glycosaminoglycans are useful with theelectrochemical test strip devices described herein, and may be usefulfor limiting, minimizing, or preventing or avoiding fouling, such as dueto the presence of non-specific proteins or substances in the testfluid. Useful charged glycosaminoglycans include, but are not limitedto, lubricin, recombinant lubricin, hyaluronic acid, heparin, heparansulfate, chondroitin sulfate, keratan sulfate, lumican, keratocan,mimecan, dermatan sulfates, glycosaminoglycans crosslinked withcollagen, or any combination of these. Optionally, the plurality ofdifferent charged glycosaminoglycans prevent electrode biofouling byrepelling non-specific proteins in the test fluid.

A variety of different electroactive redox tags are useful with theelectrochemical test strip devices described herein. For example, usefulelectroactive redox tags include, but are not limited to, methyleneblue, ferrocene or a ferrocene derivative, [Ru(NH₃)₆]^(3+/2+), anorganic metal chelate [M(L)₃]^(3+/2+) where M is Fe, Co, Os or Ru, and Lis a heterocyclic nitrogenous bidentate ligand including 2,2′-bipyridine(bipy) or 1,10-phenantroline (phen), a metalloporphyrin, an oxoosmium(VI) complex, a metalloorganic, a quinone, a hydroquinone,anthraquinone, an anthracycline, daunomycin, doxorubicin, a viologen, aphenothiazine, thionine, malachite green,2,2′-bipyridine-4,4′-dicarboxylic acid, 1,3-diaza-2-oxophenothiazine, atricyclic cytosine analog, a quinoxaline derivatives, echinomycin, orany combination of these. Optionally, the one or more electroactiveredox tags of the active capture molecules are the same as the one ormore electroactive redox tags of the null capture molecules.

In some examples, the electrochemical test strip devices include thosewhere the active capture molecule comprises one or more antibodies, oneor more monoclonal antibodies, one or more functional antibodyfragments, one or more aptamers, one or more high affinity bindingpeptides, one or more lectins, one or more receptor proteins, or one ormore enzymes. Optionally, the target analyte may be a protein, apeptide, an amino acid, an oligonucleotide, a toxin, a small molecule, asugar, a hormone, a metabolite, a biomarker, an antibody, or acombination of these. Optionally, the target analyte is a virus, a viralcomponent, a viral biomarker, a bacterial cell, a bacterial cellcomponent, a bacterial biomarker, a fungal cell, a fungal cellcomponent, a fungal biomarker, a parasite, a parasite component, aparasite biomarker, a single-celled organism, a single-celled organismcomponent, or a single-celled organism biomarker. Optionally, the testfluid is or contains saliva, nasopharyngeal fluid or mucus, pulmonaryfluid, sputum, blood, plasma, serum, whole blood, tears, urine, fecalmaterial, sweat, emesis, cervical cells or mucus, vaginal fluid ormucus, heart cells, breast milk, breast tissue cells, tumor cells,cerebrospinal fluid, skin cells, or pleural effusion.

Various different materials may be used for components of theelectrochemical test strip devices described herein. For example, one ormore of the first working electrode, the first counter electrode, thesecond working electrode, or the second counter electrode may comprisesgold, platinum, or carbon. In some examples, one or both of the firstreference electrode or the second reference electrode comprises silveror silver chloride. Advantageously, the first set of electrode contactsand the second set of electrode contacts may be configured for or usefulfor interfacing with an external reader, such as comprising an interfacedevice.

In another aspect, interface devices are disclosed, such as interfacedevices for performing potentiostatic measurements on theelectrochemical test strip devices described herein. In examples, aninterface device of this aspect comprises a set of electrode contactsfor coupling to one or more electrode contacts of an electrochemicaltest strip device; one or more potentiostats in electrical communicationwith the set of electrode contacts; and a waveform generator inelectrical or control communication with the one or more potentiostatsfor generating a potential waveform. Optionally, an interface device mayfurther comprise a processor in data or control communication with theone or more potentiostats and the waveform generator, such as aprocessor that is programmed with instructions or in data communicationwith a non-transitory computer-readable storage medium storing processorexecutable instructions. In some examples, the instructions, whenexecuted by the processor, cause the processor to perform operations.Example operations include obtaining voltammograms using the one or morepotentiostats, the waveform generator, the first set of electrodes, andthe second set of electrodes.

Optionally, the interface devices can be used to perform differentialmeasurements, using the set of electrodes. Optionally, the set ofelectrodes may be coupled to a first set of electrodes and second set ofelectrodes of an electrochemical test strip device. In some examples,the voltammograms include a test voltammogram obtained using the set ofelectrodes or a first portion thereof and a reference voltammogramobtained using the set of electrodes or a second portion thereof.Optionally, operations performed by the processor may further includedetermining a corrected voltammogram based on a difference between thetest voltammogram and the reference voltammogram. In some examples, thecorrected voltammogram provides a qualitative measure of a presence orabsence of the target analyte in the test fluid. Optionally, thecorrected voltammogram provides a quantitative measure of an amount orconcentration of the target analyte in the test fluid.

In some examples, an interface device may further comprise a button orother input device in data communication with the processor for causinginitiation of obtaining the voltammograms using the one or morepotentiostats, the first set of electrodes, and the second set ofelectrodes. The interface device may comprise an input/output device indata communication with the processor, such as a wired or wirelesscontroller, which can pass instructions to initiate obtainingvoltammograms. Optionally, the operations include receiving inputcorresponding to a directive to initiate obtaining the voltammograms.

The interface device may be configured to output a test result, such asin the form of a visible or audible signal or via data communicationwith an electronid device. In some examples, the operations includeoutputting a test result corresponding to a presence, absence, orquantitative measure of the target analyte in the test fluid based onthe voltammograms within 1-5 minutes after receiving the input.Optionally, the interface device may further one or more light emittingdiodes or other output devices in data communication with the processorfor indicating a presence, absence, or quantitative measure of thetarget analyte in the test fluid based on the voltammograms. Optionally,the operations include outputting a test result corresponding to apresence, absence, or quantitative measure of the target analyte in thetest fluid based on the voltammograms.

Advantageously, the interface device and electrochemical test stripdevices described herein can provide accurate determination of thepresence of the target analyte in the test fluid. In some examples, thetest result has a sensitivity or detection limit for the target analyteof from about 0.1 TCID₅₀/ml to about 10⁵ TCID₅₀/ml, such as from 0.1TCID₅₀/ml to 1 TCID₅₀/ml, from 1 TCID₅₀/ml to 10 TCID₅₀/ml, from 10TCID₅₀/ml to 100 TCID₅₀/ml from 100 TCID₅₀/ml to 10⁴ TCID₅₀/ml, or from10⁴ TCID₅₀/ml to 10⁵ TCID₅₀/ml. In some examples, the test result has asensitivity or detection limit for the target analyte of from about 1fg/ml to about 10 ng/ml, such as from 1 fg/ml to 10 fg/ml, from 10 fg/mlto 100 fg/ml, from 100 fg/ml to 1 pg/ml, from 1 pg/ml to 10 pg/ml, from10 pg/ml to 100 pg/ml, from 100 pg/ml to 1 ng/ml, or from 1 ng/ml to 10ng/ml. Optionally, the test result has a sensitivity or detection limitfor the target analyte of from 0.1 TCID₅₀/ml to about 10 TCID₅₀/ml or asensitivity or detection limit for the target analyte of from about 1fg/ml to about 1 pg/ml.

The interface devices useful with the electrochemical test strip devicesdescribed herein may have any suitable physical configuration. In someexamples, the interface device comprises or is constructed as a cable,adapter, or electrochemical test strip reader coupleable to asmartphone, tablet, PC, electronic device, or universal serial bus (USB)device or port. Optionally, the interface device comprises or isconstructed as a bluetooth or wireless electrochemical test strip readercoupleable to a smartphone, tablet, PC, or electronic device.

In another aspect, kits are provided herein, such as electrochemicaltest strip kits. In some examples, a kit of this aspect may comprise anelectrochemical test strip device (e.g., any of the electrochemical teststrip devices described herein) and instructions for applying a testfluid to the test chamber and for coupling the electrochemical teststrip device to an interface device to obtain a test result indicating apresence, absence, or quantitative measure of the target analyte in thetest fluid. Optionally, kits of this aspect may further comprise aninterface device for coupling to the electrochemical test strip device(e.g., any of the interface devices described herein).

In another aspect, systems are provided herein, such as systems forperforming electrochemical assays. In examples, a system of this aspectcomprises an electrochemical test strip device (e.g., any of theelectrochemical test strip devices described herein); and an interfacedevice for coupling to the electrochemical test strip device (e.g., anyof the interface devices described herein).

In other aspects, methods are described herein, such as methods forperforming electrochemical assays. In some examples, a method of thisaspect comprises providing an electrochemical test strip device (e.g.,any of the electrochemical test strip devices described herein),applying the test fluid to the fluid chamber; coupling theelectrochemical test strip device to an interface device (e.g., any ofthe electrochemical test strip devices described herein); and obtainingvoltammograms using the electrochemical test strip device and/or theinterface device. Optionally, the voltammograms include a testvoltammogram obtained using the first set of electrodes of theelectrochemical test strip device and a reference voltammogram obtainedusing the second set of electrodes the electrochemical test stripdevice.

In some examples, methods of this aspect may further include determininga corrected voltammogram based on a difference between the testvoltammogram and the reference voltammogram. Optionally, the correctedvoltammogram provides a qualitative measure of a presence or absence ofthe target analyte in the test fluid. Optionally, the correctedvoltammogram provides a quantitative measure of an amount orconcentration of the target analyte in the test fluid. Methods of thisaspect may further comprise outputting a test result corresponding to apresence, absence, or quantitative measure of the target analyte in thetest fluid based on the voltammograms. Advantageously, outputting thetest result may occurs within 1-5 minutes of initiating obtaining thevoltammograms or in less than 1 minute, less than 2 minutes, less than 3minutes, less than 4 minutes, or less than 5 minutes.

In some examples, the test result has a sensitivity or detection limitfor the target analyte of from about 0.1 TCID₅₀/ml to about 10⁵TCID₅₀/ml, such as from 0.1 TCID₅₀/ml to 1 TCID₅₀/ml, from 1 TCID₅₀/mlto 10 TCID₅₀/ml, from 10 TCID₅₀/ml to 100 TCID₅₀/ml from 100 TCID₅₀/mlto 10⁴ TCID₅₀/ml, or from 10⁴ TCID₅₀/ml to 10⁵ TCID₅₀/ml. In someexamples, the test result has a sensitivity or detection limit for thetarget analyte of from about 1 fg/ml to about 10 ng/ml, such as from 1fg/ml to 10 fg/ml, from 10 fg/ml to 100 fg/ml, from 100 fg/ml to 1pg/ml, from 1 pg/ml to 10 pg/ml, from 10 pg/ml to 100 pg/ml, from 100pg/ml to 1 ng/ml, or from 1 ng/ml to 10 ng/ml. Optionally, the testresult has a sensitivity or detection limit for the target analyte offrom 0.1 TCID₅₀/ml to about 10 TCID₅₀/ml or a sensitivity or detectionlimit for the target analyte of from about 1 fg/ml to about 1 pg/ml.

Without wishing to be bound by any particular theory, there can bediscussion herein of beliefs or understandings of underlying principlesrelating to the invention. It is recognized that regardless of theultimate correctness of any mechanistic explanation or hypothesis, anembodiment of the invention can nonetheless be operative and useful.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and FIG. 1B provide schematic illustrations of an exampleelectrochemical test strip device.

FIG. 2A shows a top-view schematic illustration of an exampledual-chamber electrochemical test strip device with a top fillconfiguration. FIG. 2B shows a top-view schematic illustration of anexample dual-chamber electrochemical test strip device with an edge fillconfiguration.

FIG. 3 shows a schematic illustration of an electrochemical test stripdevice connected to an interface device.

FIG. 4 shows a schematic illustration of a working electrode of anexample electrochemical test strip device functionalized with capturemolecules for use in a biosensor rapid antigen assay.

FIG. 5 shows a schematic illustration of a working electrode of anexample electrochemical test strip device functionalized for anelectrochemical biosensor based antibody assay to determine host immuneresponse.

FIG. 6 shows a schematic illustration of a working electrode of anexample electrochemical test strip device functionalized for anelectrochemical biosensor rapid host immune response assay.

FIG. 7 shows a schematic illustration of a working electrode of anexample electrochemical test strip device functionalized for anelectrochemical biosensor rapid host immune response assay.

FIG. 8 shows a schematic illustration of a bioprinted electrochemicalbiosensor based multi-antigen assay using influenza and SARS-CoV-2 asexamples.

FIG. 9 provides a schematic illustration of another electrochemicalbiosensor rapid antigen assay exemplified as a functionalizedelectrochemical test strip device.

FIG. 10 provides a schematic illustration of another electrochemicalbiosensor rapid antigen assay exemplified as a functionalizedelectrochemical test strip device.

FIG. 11 provides a schematic illustration of another electrochemicalbiosensor rapid antigen assay exemplified as a functionalizedelectrochemical test strip device.

FIG. 12 provides a schematic illustration of another electrochemicalbiosensor rapid antigen assay exemplified as a functionalizedelectrochemical test strip device.

FIG. 13 provides an overview of an example interface device fordetecting analytes using an electrochemical test strip device.

FIG. 14 shows an illustration of an example handheld interface device.

FIG. 15A and FIG. 15B show illustrations of a mobile electronic devicewith an interface device connected to a port of the mobile electronicdevice. The interface device is depicted as an adapter-format test stripreader in FIG. 15A and as a cable-based test strip reader in FIG. 15B.

FIG. 16 provides an overview of a wide-scale pandemic Real-Time TestingSystem (RTTS).

FIG. 17A provides voltammogram data obtained using an electrochemicaltest strip device functionalized with SARS-CoV-2 S1 monoclonalantibodies as capture molecules. FIG. 17B provides a dose response curvewhen the immobilized S1 monoclonal antibody was tested againstrecombinant S1+S2 ECD spike antigens.

FIG. 18 provides voltammogram data obtained using an electrochemicaltest strip device functionalized with SARS-CoV-2 antibodies as capturemolecules tested against SARS-CoV-2 spike antigens vs influenzaneuraminidase antigens as test analytes.

FIG. 19A provides voltammogram data obtained using an electrochemicaltest strip device functionalized with SARS-CoV-2 S1 monoclonalantibodies as capture molecules. FIG. 19B provides a dose response curvewith standard deviations when the immobilized S1 monoclonal antibody wastested against wild-type heat inactivated viruses in buffer.

FIG. 20 provides data showing the current output obtained using anelectrochemical test strip functionalized with an active capturemolecule and tested using saliva as a test fluid with buffer solution asa reference to investigate biofouling.

FIG. 21 provides data showing the current output with increasingconcentrations of heat inactivated virus in pooled saliva when usingelectrochemical test strips modified with a biofouling preventative.

FIG. 22 provides data from a blind study that was performed usingcontrived human saliva samples.

FIG. 23A provides voltammogram data and FIG. 23B provides current outputdata obtained using an electrochemical test strip device functionalizedwith SARS-CoV-2 S1 monoclonal antibodies as capture molecules testedagainst SARS-CoV-2 variant antigens with different mutations.

FIG. 24 provides voltammogram data obtained using an electrochemicaltest strip device functionalized with methylene blue tagged ACE2proteins as capture molecules.

FIG. 25 provides voltammogram data from a validation test obtained usingan electrochemical test strip device functionalized with methylene bluetagged ACE2 proteins as capture molecules.

FIG. 26 provides voltammogram data from another validation testperformed to determine if the receptor binding domain (RBD) ofSARS-CoV-2 antigens are active.

FIG. 27A provides voltammogram data and FIG. 27B provides dose responsecurve data where ACE2 protein was immobilized on the working electrodeof an electrochemical test strip that was evaluated for binding with RBDantigens at different concentrations.

FIG. 28A provides voltammogram data and FIG. 28B provides current outputdata obtained using an electrochemical test strip device functionalizedwith ACE2 protein as capture molecules tested against SARS-CoV-2 variantantigens with different mutations.

FIG. 29 provides comparative data showing differences in bindingaffinity based on using different capture molecules.

FIG. 30 provides comparative data showing differences in bindingaffinity based on using different capture molecules.

DETAILED DESCRIPTION

The present disclosure provides devices, systems, kits, methods, andtechniques for analyzing test fluids to determine presence, absence, orconcentration of analytes in the test fluids and are useful forperforming diagnostic testing, such as to quickly identify whether ornot an individual may have a particular disease or condition. Thedevices, systems, kits, methods, and techniques described herein can beused to detect virtually any analyte of interest using an appropriatelyfunctionalized electrochemical test strip. The electrochemical teststrip can be evaluated using an interface device that couples withelectrode contacts of the electrochemical test strip to measureelectrochemical data indicating presence, absence, or concentrations ofthe analytes of interest, allowing for prompt identification of whetheror not an individual has a particular disease or condition, such as in aperiod of 5 minutes or less.

The test fluid can comprise any suitable body fluid or body materialdissolved or suspended in a liquid, which can be used in diagnosing anindividual for a particular disease or condition. Non-limiting exampletest fluids may include or comprise saliva, nasopharyngeal fluid ormucus, pulmonary fluid, sputum, blood, plasma, serum, whole blood,tears, urine, fecal material, sweat, emesis, cervical cells or mucus,vaginal fluid or mucus, heart cells, breast milk, breast tissue cells,tumor cells, cerebrospinal fluid, skin cells, or pleural effusion.

The analytes may be infections particles (e.g., virus, bacteria, etc.)or components thereof (e.g., proteins, nucleic acid sequences, etc.) orbiomarkers (e.g., inflammatory biomarkers, antigens, antibodies, hostproteins, small molecules, metabolites, toxins, etc.) indicative of aparticular disease or condition. As some non-limiting examples, thedisease or condition may be a viral infectious disease, a cardiacdisease or condition, a renal disease or condition, a hepatic disease orcondition, cancer, a neurodegenerative disease or condition, a sexuallytransmitted disease, ingestion of illicit substances, or ingestion ofperformance-enhancing substances. Depending on the analytes of interest,particular body fluids may be more suitable for detection than otherfluids. For example, in the case of the target analyte being arespiratory virus, saliva or nasopharyngeal fluid or mucus may be auseful test fluid. As another example, in the case of the target analytebeing a metabolite, urine may be a useful test fluid.

The test fluid can also or alternatively be an environmental fluid,which can be used to diagnose the presence, absence, or concentration ofanalytes of interest in the environment. In some non-limiting examples,the test fluid can be groundwater, river water, sea water, processwater, waste water, reactor effluent, gaseous effluent. In some cases, agaseous fluid can be dissolved in water or an organic solvent. As somenon-limiting examples, analytes of interest include contaminants,organics, heavy metals, toxins, small molecules, bacteria, bacterialcomponents, viral particles, viral components, or the like.

The electrochemical test strips used in the devices, systems, kits,methods, and techniques described herein can employ an electrodefunctionalized with capture molecules that can bind the analytes ofinterest. The capture molecules can include, for example, antibodies,antigens, aptamers, or the like, which are specifically structured orotherwise configured to bind the analytes of interest. The capturemolecules can include electroactive redox tags (also referred to asredox-active molecules) that can undergo electrochemical oxidationand/or reduction in an electrochemical detection scheme, and allow foridentification of the presence of the analytes of interest. In somenon-limiting examples, the electroactive redox tags may comprisemethylene blue, ferrocene or a ferrocene derivative, [Ru(NH₃)₆]^(3+/2+),an organic metal chelate [M(L)₃]^(3+/2+) where M is Fe, Co, Os or Ru,and L is a heterocyclic nitrogenous bidentate ligand including2,2′-bipyridine (bipy) or 1,10-phenantroline (phen), a metalloporphyrin,an oxoosmium (VI) complex, a metalloorganic, a quinone, a hydroquinone,anthraquinone, an anthracycline, daunomycin, doxorubicin, a viologen, aphenothiazine, thionine, malachite green,2,2′-bipyridine-4,4′-dicarboxylic acid, 1,3-diaza-2-oxophenothiazine, atricyclic cytosine analog, a quinoxaline derivatives, echinomycin, orany combination of these.

In examples, the electroactive redox tags can be part of, bonded to, orotherwise attached to the capture molecules bound to one or moreelectrodes of an electrochemical test strip. When the capture moleculesbind the target analyte, a distance between the electroactive redox tagand the electrode can be modulated from the distance in the unboundstate, providing a measurable electrical feedback or signal indicatingbinding of the capture molecules to the target analyte. The measurablesignal can allow for very sensitive detection of extremely smallconcentrations of target analytes, such as on the femtogram/ml scale. Inthe case of infectious viral particles, detections as small as 0.1TCID₅₀/ml are achievable.

The presently disclosed devices, systems, kits, methods, and techniquesemploy electrodes functionalized with capture molecules, which can beprepared as self-assembled-monolayers (SAMs) or mixed SAMs. As referredto herein, a mixed SAM is a SAM including multiple monolayer components,as opposed to a SAM including just a single component making up theentirety of the monolayer. The capture molecules can be attached to theelectrode by way of a linker component that terminally binds or isterminally bonded to the capture molecules. As a non-limiting example,the linker component may comprise a thiolated polyethylene glycol (PEG)group, which can form a sulfur-metal bond to metal surfaces (e.g., gold,silver, copper, platinum, etc.) to generate a self-assembled monolayer.

Partial coverage of the electrode surface with the capture molecules maybe sufficient and so passivation components that do not include thecapture molecules can be included in the SAM. For example, a mixed SAMmay include a linker component (e.g., binding or bonded to the capturemolecule) and a passivation component, which may optionally be charged.In some examples, the passivation component may be a thiolated PEG groupthat is not bound to the active capture molecules. Optionally, thepassivation component may include one or more carboxylic acid groups,which can be deprotonated to form a charged passivation component. Insome examples, the surface of the electrode can be functionalized with amixed SAM by exposing the electrode to a solution containing a mixtureof different thiolated PEG molecules, such as including a first PEGmolecule that comprises one or more carboxylic acid groups (e.g., as acharged passivation component) and a second PEG molecule includingdifferent functionality (e.g., to bond to or bind the capture molecule).

In some examples, click chemistry can be used to couple or bond thecapture molecule to a linker component. Click chemistry can be extremelyspecific and allow coupling of two different molecules in an adduct orbioconjugate configuration precisely with limited side reactions. Insome examples, click chemistry can include bond-forming reactionsinvolving azido functionalized molecules and alkyne functionalizedmolecules. In some examples, use of alkyne-terminated PEG molecules canbe useful for coupling the capture molecules functionalized with azidogroups to the electrode using click chemistry. Optionally, use of azidofunctionalized PEG molecules can be useful for coupling the capturemolecules functionalized with alkyne groups to the electrode using clickchemistry.

In the case of mixed SAMs, a first component of the SAM can includealkyne-terminated PEG-thiol molecules and a second component of the SAMcan include PEG-thiol molecules that are not functionalized with alkynegroups, such as including non-functionalized PEG-thiol molecules orcarboxylic acid-functionalized PEG-thiol molecules. In this way, onlypart of the SAM will include alkyne-terminated PEG-thiol molecules thatcan be linked to the capture molecules using click chemistry, asdescribed above. Advantageously, such mixed-SAMs can be preparedefficiently using thiol surface linking by exposing an electrode surfaceto a mixture of alkyne-terminated PEG-thiol molecules and PEG-thiolmolecules without alkyne termination. In some cases, a mixed-SAM can beprepared in time periods as short as from about 1 hour to about 5 hours,such as from 1 hour to 1.5 hours, from 1.5 hours to 2 hours, from 2hours to 2.5 hours, from 2.5 hours to 3 hours, from 3 hours to 3.5hours, from 3.5 hours to 4 hours, from 4 hours to 4.5 hours, or from 4.5hours to 5 hours.

The surface-bound alkyne-terminated PEG thiol molecules can be subjectedto reaction with azido-functionalized capture molecules to functionalizean electrode with the capture molecules (e.g., by click chemistry).Optionally, the reaction can proceed by exposing the mixed SAM includingalkyne-terminated PEG-thiol molecules and azido-terminated PEG-thiolmolecules to the azido-functionalized capture molecules, such as whileheating and/or in the presence of a catalyst (e.g., a copper catalyst).In some examples, the reaction can functionalize the surface withcapture molecules in time periods as short as from about 1 hour to about5 hours, such as from 1 hour to 1.5 hours, from 1.5 hours to 2 hours,from 2 hours to 2.5 hours, from 2.5 hours to 3 hours, from 3 hours to3.5 hours, from 3.5 hours to 4 hours, from 4 hours to 4.5 hours, or from4.5 hours to 5 hours. Accordingly, the preparation of the mixed SAM andfunctionalization with capture molecules can be completed rapidly, suchas in from about 2 hours to about 10 hours, for example, from 2 hours to2.5 hours, from 2.5 hours to 3 hours, from 3 hours to 3.5 hours, from3.5 hours to 4 hours, or from 4 hours to 4.5 hours, from 4.5 hours to 5hours, from 5 hours to 5.5 hours, from 5.5 hours to 6 hours, from 6hours to 6.5 hours, from 6.5 hours to 7 hours, from 7 hours to 7.5hours, from 7.5 hours to 8 hours, from 8 hours to 8.5 hours, from 8.5hours to 9 hours, from 9 to 9.5 hours, or from 9.5 hours to 10 hours. Insome cases, preparation of the mixed SAM functionalization can becompleted in less than 6 hours.

Although the electrochemical test strips can be very sensitive to targetanalytes, in some cases they can also be sensitive to other analytes,molecules, or components that can interact with the electrodes, theelectroactive redox tags, or the capture molecules, which can cause bothfalse positive and false negative detections. Such interference ofdetection of target analytes by non-target analytes or other test fluidcomponents may be referred to herein as fouling or biofouling (e.g., inthe case of interferents originating from biological materials or fluidswhich may be present in the test fluid). Advantageously, the presentlydisclosed devices, systems, kits, methods, and techniques can limitfouling or biofouling and provide sensitive specific detection of targetanalytes even when the test fluid includes other analytes, molecules, orcomponents. As an example, an anti-fouling hydrogel may be coated overor with the mixed SAM, such as a hydrogel comprising one or more chargedglycosaminoglycans. In some cases a plurality of different chargedglycosaminoglycans can be used. Non-limiting examples of chargedglycosaminoglycans include lubricin (e.g., recombinant lubricin),hyaluronic acid, heparin, heparan sulfate, chondroitin sulfate, keratansulfate, lumican, keratocan, mimecan, dermatan sulfates,glycosaminoglycans crosslinked with collagen. By using a hydrogelincluding different charged glycosaminoglycans coated over or with amixed SAM including a passivation component (e.g., a charged passivationcomponent) and a linking component terminally binding a capturemolecule, sensitive and specific detection of target analytes can beachieved. Sensitivity can be further improved by simultaneous use of areference electrode system constructed essentially similarly, butincluding capture molecules that do not bind the target analyte (e.g.,null capture molecules), instead of capture molecules that do bind thetarget analyte (e.g., active capture molecules).

Electrochemical Test Strip Devices

FIG. 1A and FIG. 1B provide schematic illustrations of an exampleelectrochemical test strip device 100. FIG. 1A shows a top-down view ofelectrochemical test strip device 100 and FIG. 1B shows a perspectiveview of electrochemical test strip device 100. Electrochemical teststrip device 100 includes a fluid chamber 105 containing two sets ofthree electrodes. Fluid chamber 105 includes a sensing chamber 105 a (ortest chamber) and a reference chamber 105 b (or control chamber), eachwith a corresponding set of three electrodes. In some cases, sensingchamber 105 a and reference chamber 105 b can be physically separatedfrom each other (e.g. by a barrier) or they can each be a subset offluid chamber 105 in contact with a corresponding set of threeelectrodes. Electrochemical test strip device 100 also includes aholding or grasping area 110, which may optionally include a label,identifier, or barcode, which can facilitate identification of theparticular analyte the electrochemical test strip device 100 isconfigured to detect, or other information. In some cases, holding orgrasping area 110 is optional, and an identifier may optionally bepositioned elsewhere on electrochemical test strip device 100.Electrochemical test strip device 100 also includes a cover plate 115with an opening 120 for directing a test fluid to the fluid chamber 105,and a set of contacts 125 to enable electrical connection to aninterface device. The three electrodes in each of the testing chamber105 a and the reference chamber 105 b support stimulus and measurementvia a potentiostat approach. In this approach, a fixed voltage isapplied between two of the electrodes. The third electrode completes acircuit to enable precise measurement of current flow at those voltageconditions. Different voltages can be applied, in sequence, and thecurrent at each voltage measured to produce a voltammogram, providingcurrent as a function of voltage. The three electrodes are called thereference electrode (RE), working electrode (WE), and counter electrode(CE). Specifically, testing chamber 105 a is depicted as including areference electrode 130 a, a working electrode 135 a, and a counterelectrode 140 a and reference chamber 105 b is depicted as including areference electrode 130 b, a working electrode 135 b, and a counterelectrode 140 b.

Holding or grasping area 110 can comprise an extended region of a basesupporting the two sets of electrodes and can comprise any suitablenon-conducting material, such as a plastic, polymeric, fibrous, ornon-porous material. The two sets of electrodes can comprise anysuitable electrode material. For use in potentiostatic measurements, itmay be beneficial for reference electrodes 130 a and 130 b to compriseAg/AgCl, and for working electrodes 135 a and 135 b and counterelectrodes 140 a and 140 b to comprise another metal, such as Au, Ag,Cu, or Pt. The legs of the electrodes may extend to the set of contacts125 as the same conductive material present in fluid chamber 105, or theconductive material may change at the border of or outside fluid chamber105 to another conductive material. Cover plate 115 can comprise anysuitable non-conducting material, such as a plastic, polymeric, fibrous,or non-porous material.

In this example, working electrode 135 a can be functionalized with acapture molecule, such as an active capture molecule that binds aparticular analyte of interest. In some examples, the capture moleculecan be an antibody or antibody component, functionalized with anelectroactive redox tag, and coupled to the surface of the workingelectrode 135 a by click chemistry, as described above, such as by wayof a linking component (e.g., PEG-thiol molecule). The working electrode135 a can also include a charged passivation component (e.g., chargedPEG-thiol molecules) that do not include the capture molecule. Theworking electrode 135 b can be functionalized with a different capturemolecule, such as a null capture molecule that does not binds theparticular analyte of interest or that binds some other analyte. In someexamples, the null capture molecule can be an antibody or antibodycomponent, functionalized with an electroactive redox tag, and coupledto the surface of the working electrode 135 b by click chemistry, asdescribed above, such as by way of a linking component (e.g., PEG-thiolmolecule). The working electrode 135 b can also include a passivationcomponent (e.g., a charged passivation component, such as chargedPEG-thiol molecules) that do not include the capture molecule. Inaddition, working electrodes 135 a and 135 b can be coated, at leastpartly, with a hydrogel comprising a plurality of different chargedglycosaminoglycans.

FIG. 2A shows a top-view schematic illustration of an exampledual-chamber electrochemical test strip device 200 with a top fillconfiguration. In this example, there are two separate measurementchambers, a test chamber 205 a with a reference electrode 230 a, aworking electrode 235 a, and a control electrode 240 a, and referencechamber 205 b with a reference electrode 230 b, a working electrode 235b, and a control electrode 240 b. Two microfluidic channels areconnected to the collection well 205 to bring the sample from there tothe test chamber 205 a and reference chamber 205 b. The test chamber 205a is functionalized, with an attached antibody, such as at the workingelectrode 205 a, while the reference chamber is not functionalized inthe same way.

FIG. 2B shows a top-view schematic illustration of an exampledual-chamber electrochemical test strip device 250 with an edge fillconfiguration. In this example, there are two separate measurementchambers, a test chamber 255 a with a reference electrode 280 a, aworking electrode 285 a, and a control electrode 290 a, and referencechamber 255 b with a reference electrode 280 b, a working electrode 285b, and a control electrode 290 b. Two microfluidic channels areconnected to the collection well 255 to bring the sample from there tothe test chamber 255 a and reference chamber 255 b. The test chamber 255a is functionalized, with an attached antibody, such as at the workingelectrode 255 a, while the reference chamber is not functionalized inthe same way.

FIG. 3 shows a schematic illustration of an electrochemical test stripdevice 300 connected to an interface device 350. Interface device 350comprises potentiostat circuit, which may comprise a variety ofelectrical circuit components including operational amplifiers(op-amps), control muxes, and precision resistors and capacitors, forexample. The op-amps and muxes can themselves be built from standardMOSFETs, which are a form of transistors easily fabricated intointegrated circuits. The potentiostat circuit may be implemented as asingle-chip integrated circuit, enables the interface device 350 to besmall, so that it can be easily hand-held. In some examples, thecircuits components of the potentiostat circuit are analog in nature,but these can be coupled to a digital, general purpose microprocessor ormicrocontroller. The microprocessor can specify the input voltage to useand analyze the output signals. To enable this interaction, theinterface device an also include very accurate digital to analogconverters (DACs) and analog to digital converters (ADCs). In theconfiguration illustrated in FIG. 3 , two separate potentiostat circuitsare used to connect to the different sets electrodes of the sensingchamber and test chamber of electrochemical test strip device 300 toprovide a differential signal, determined by a differential amplifier,that is converted from an analog signal to a digital signal and providedto the microcontroller. When an analyte binds to a capture moleculetethered to the working electrode, the current detected by thepotentiostat circuit can be modulated (e.g., reduced) at a particularvoltage, allowing detection of the binding event. Without wishing to bebound by any theory, the modulation in current can be due to adifference in proximity between an electroactive redox tag component ofthe capture molecule on binding the analyte that may occur due to anincreased hydrodynamic drag due to the presence of the bound analyte.Generally, when an analyte is bound to the capture molecule, a reductionin current can be observed at a particular voltage compared to thecurrent observed when no analyte is bound.

The disclosed electrochemical test strip devices and interface devicescan be used together to sensitively and quickly determine the presence,absence, or concentration of a particular analyte in a test fluid foruse in diagnosing a condition, infection, or disease. In some examples,any of a variety of analytes may be detected by functionalizing aworking electrode of the electrochemical test strip device with anappropriate capture molecule. Conditions, infections, or diseases ofinterest include, but are not limited to: SARS-CoV2, SARS-CoV2 variant,Influenza, Ebola, Malaria, Acute coronary syndrome, Acute Myocardialinfarction, Heart failure, Cardiac Ischemia, Acute Coronary Syndrome,Acute Cardiac Disorders, Transplant rejection, Schistosomiasis,Lymphatic filariasis, Onchocerciasis, Chagas Disease, Africantyrpanosomaniasis, Leishmaniasis, Leprosy, Dengue Fever, Tuberculosis,Hookworm, Trichuriasis, Treponematoses, Buruli Ulcer, Dracunculiasis,Leptospirosis, Strongyloidiasis, Foodborne trematodiases,Neurocysticercosis, Flavivirus, Stroke, Traumatic Brain Injury, Livercancer and germ cell tumors, Multiple myeloma, chronic lymphocyticleukemia, lymphomas, Choriocarcinoma and germ cell tumors, Thymoma,Thyroid carcinoma, Testicular germ cell tumor/carcinoma, Ovarian germcell tumor/carcinoma, Saccrococcygeal teratoma, Bladder cancer andcancer of the kidney or ureter, Colorectal cancer, Neuroendocrinetumors, Lung cancer, pancreatic cancer, Gastrinoma, Small cell lungcancer and neuroblastoma, Metastatic prostate cancer, Mesothelioma,Thyroid cancer, Kidney schlerosis, kidney fibrosis, glomerular injury,kidney failure, glomerulosclerosis, or glomerulonephritis, Acute Kidneyinjury, Interstitial cystitis, Kidney Graft rejection, Illicit drug use,performance enhancing substance use, Syphilis, Gonorrhea, Chlamydia,Trichomoniasis, HBV, HSV, HIV, HCV, HPV, MRSA, Zika, Sepsis, Cytokinestorm, Acute pulmonary embolism, Heart Failure, Nonalcoholic fatty liverdisease, Pancreatitis, Neurodegeneration, brain injury, Alzheimer's,Parkinson's, or combinations of these.

Analytes of interest for detecting or diagnosing the above diseases,infections, or conditions include, but are not limited to: SARS-CoV-2nucleocapsid protein, SARS-CoV-2 Spike protein, influenza high affinityhemagglutinin binding protein, Ebola virus Glycoprotein, ANG-2, VWF,VWFpp, slCAM-1, sFLT-1, sTie-2, CRP, PCT, IP-10, CHI3L1 and sTREM-1,Cardiac Troponin I (cTnI), H-FABP, BNP, NT Pro-BNP, Nitrated cTnI,acetate, acetone, cytosine, methylmalonate, phenylacetylglycine, solubleCD40 ligand, atrial natriuretic peptide (ANP), schistosome circulatingcathodic antigen, Circulating antigens detected by OG4c3 Mab, Oncho-C27antigen, Trypanosoma cruzi circulating antigens, GM6, Leishmania majorantigen TSA, phenolic glycolipid-I antigen of M. leprae, Dengue NS 1and/or envelope protein, RD1, RD2, or RD3 of Mycobacterium tuberculosis,hookworm ASPS, Whipworm coproantigen, Tp47, Tp17, Tp15, TmpA, or TmpB,mycolactone, the primary lipidic toxin A of Mycobacterium ulcerans,OV-10, OV-11 and OV-16, core lipopolysaccharide of Leptospira spp,Strongyloidiasis spp L3 stage antigens, secretory coproantigens ofintestinal and liver flukes, T24, NS5, S100 calcium binding protein B(S-100B), neuron-specific enolase (NSE), myelin basic protein (MBP),Microtubule Associated Protein 2 (MAP2), glial fibrillary acidic protein(GFAP), ubiquitin carboxyl-terminal esterase L1 (UCH-L1), glialfibrillary acid protein (GFAP), aldehyde dehydrogenase 1 family memberLI (ALDHILI), phosphorylated neurofilament heavy chain (pNFH), mediumchain (NFM), or light chain (NFL), alpha-synuclein, visinin-like protein1 (VILIP-1), S100B, Neuron Specific Enolase (NSE), Glial FibrillaryAcidic Protein (GFAP), Ubiquitin Carboxy-Terminal Hydrolase LI (UCH-L1),Interleukin-1b (IL-Ib), Interferon Gamma (IFN-g), Interleukin 8 (IL-8),Interleukin 10 (IL-10), Spectrin II, 8-Hydroxy-2′-Deoxyguanosine(8-OHdG), Alpha-fetoprotein (AFP), Beta-2-microglobulin (B2M),Beta-human chorionic gonadotropin (Beta-hCG), Bladder Tumor Antigen(BTA), Carcinoembryonic antigen (CEA), epidermal growth factor receptor,leucine-rich alpha-2 glycoprotein, inter-alpha trypsin inhibitor heavychain 3, inter-alpha trypsin inhibitor heavy chain 4, dipeptidylpeptidase 4, peptidase inhibitor 16, coagulation factor V, C-reactiveprotein, Rho-GDP dissociation inhibitor 1 isoform A, hemopexin,extracellular superoxide dismutase[Cu—Zn], thrombospondin-4, collagenalpha-1(1) chain, cadherin-2, vitronectin, Chromogranin A (CgA),Cytokeratin fragment 21-1, Gastrin, Neuron-specific enolase (NSE),Nuclear matrix protein 22, Prostate specific antigen (PSA), C reactiveprotein (CRP), chromogranin A (CHGA), bone alkaline phosphatase (BAP),cysteine-rich secretory protein 3 (CRISP3), ERG, prostatic acidphosphatase (PAP; also called ACP3; human acid phosphatase 3,prostatic), Apolipoprotein A2 (ApoA2), Apolipolipoprotein C1 (ApoC1),Soluble mesothelin-related peptides (SMRP), Thyroglobulin, S100A8, CSTA(cystatin A), GRM1 (glutamate receptor, metabotropic 1), TPT1 (tumorprotein, translationally-controlled 1), GRIK1 (glutamate receptor,ionotropic, kainate 1), H6PD (hexose-6-phosphate dehydrogenase), IGF2BP1(insulin-like growth factor 2 mRNA binding protein 1), MDM4 (3T3 celldouble minute 4), CA6 (carbonic anhydrase VI), epidermal growth factorreceptor, leucine-rich alpha-2 glycoprotein, inter-alpha trypsininhibitor heavy chain 3, inter-alpha trypsin inhibitor heavy chain 4,dipeptidyl peptidase 4, peptidase inhibitor 16, coagulation factor V,C-reactive protein, Rho-GDP dissociation inhibitor 1 isoform A,hemopexin, extracellular superoxide dismutase[Cu—Zn], thrombospondin-4,collagen alpha-1(1) chain, cadherin-2, vitronectin, Annexin A1, Rab23,Kidney injury molecule-1 (KIM-1), neutrophil gelatinase associatedlipocalin (NGAL), interleukin-18 (IL-18), hepatocyte growth factor(HGF), cystatin C (Cys), N-acetyle-β-D-glucosaminidase (NAG), vascularendothelial growth factor (VEGF), chemokine interferon-inducible protein10 (IP-10; CXCL10), Antiproliferative factor (APF), CD44, UMOD, PEDF,MMPI, SERPING1, COL1A2, COL3A1, TIMP1, neurogranin (NRGN), myelin basicprotein (MBP), glial fibrillary acid protein (GFAP), peptidylargininedeiminase (PAD), IGF-I, IGF-II, Aβ-40, Aβ-42, alpha amylase, IL-1 beta,TNF-alpha, Tp47, Tp17, Tp15, TmpA, TmpB, Gonorrhea antigens, Chlamydiaantigens, Trichomonas adhesin peptide, PreS1, PreS2, gC2, gG2, gB2, gD2,P24, Oxycodone, opiates, cocaine, cannabinoids, barbiturates,methamphetamine, amphetamine, benzodiazepine, methadone, TCAs, ecstasy,cotinine, Clenbuterol, DHEA, 7-hydroxy-DHEA, 7-Keto-DHEA(dehydroepiandrosterone), andarine, ostarine, LGD-4033, RAD-140,albuterol, formoterol, salmeterol, androstenedione, methenolone,Tetrahydrogestrinone, oxymetholone, Oxandrolone, methandrostenolone,Stanozolol, nandrolone decanoate, nandrolone phenpropionate,testosterone cypionate, boldenone undecylenate, Testosterone enanthate,Epogen, Procrit, hCG, anastrozole, tamoxifen, P24, c200-3, NS-5 and amodified core antigen, epitope of the C-terminal or the N-terminalregion of a HPV E7 protein, MV0118 protein, lateral ridge (LR) or the CCloop within domain III (DIM) of the E protein of Zika virus,Procalcitonin, Angiopoietin 1 and 2, Endocans, TREM-1, ctla-4,Staphylococcus aureus, Coagulase-negative staph, Streptococcuspneumonia, Haemophilus influenza b, Neisseria meningitis, Klebsiellapneumonia, Enterococcus faecalis, Acinetobacter baumanii, Escherichiacoli, Salmonella enterica, Shigella dysenteriae, Citrobacter freundii,Serratia marcescens, Proteus mirabilis, Pseudomonas aeruginosa,Bacteroides fragilis IL-2, IL-6, IL-7, IL-10, G-CSF, TNF, CXCL10, MCP1,MIP1a, D-Dimer, C-reactive protein, LDH, ferritin, IFN-γ, IP-10, IL-6,MCP-1, TNF-α, IgA, IgG, IgM, Procalcitonin (PCT), C-reactive protein(CRP), Interleukin 6 (IL-6), Pre-sepsin, Soluble intercellular adhesionmolecule-1 (sICAM-1), Endocan, D-dimer, Lactate dehydrogenase (LDH), orcombinations or metabolites thereof. In some examples, particularanalytes may be useful for diagnosing a severity of an infection, suchas infection by SARS-CoV-2 or a SARS-CoV-2 variant, such asProcalcitonin (PCT), C-reactive protein (CRP), Interleukin 6 (IL-6),D-dimer, Lactate dehydrogenase (LDH).

FIG. 4 shows a schematic illustration of a working electrode 400 of anexample electrochemical test strip device functionalized with capturemolecules for use in a biosensor rapid antigen assay. In this example,the capture molecules include antibodies that bind SARS-CoV-2. Theworking electrode 400 comprises gold screen printed on a surface. Cuassisted click chemistry is used to covalently link azido 405 modifiedmonoclonal SARS-CoV-2 antibodies 410 with an electroactive redox tag 415to the gold surface via a PEG-thiol linker with a terminal alkyne group420. An anti-biofouling layer made of a hydrogel comprising charged PEGlayers 425 and lubricin (PRG4) 430 helps repel the non-specific bindingproteins in the sample. This virus-detecting assay uses the kineticresponse of a probe/virus complex to analyze the complexation state ofthe antibody. This approach employs electrode-tethered sensors includingan analyte-binding antibody 410 tagged with an electroactive redox probe415 covalently bound on a neutrally charged PEG linker. When a positivepotential is applied to the working electrode 400, the proximity betweenthe electroactive redox tag 415 and the gold surface is modulated. Thebinding between the antibodies 410 and the viral proteins 435 of theSARS-CoV-2 viral particles 440 induces a hydrodynamic drag on themodulation, allowing the presence of viral proteins 435 and particles440 to be detected using potentiometry measurements.

FIG. 5 shows a schematic illustration of a working electrode 500 of anexample electrochemical test strip device functionalized for anelectrochemical biosensor based antibody assay to determine host immuneresponse, again using SARS-CoV-2 as an example. The working electrode500 comprises gold screen printed on a surface. Cu assisted Clickchemistry is used to covalently link azido 505 modifiedanti-immunoglobulin antibodies 510, 515, and/or 520, including anelectroactive redox tag 525, to the gold surface via a PEG-thiol linkerwith a terminal alkyne group 530. This allows preparation ofelectrode-tethered anti-antibodies 510, 515, and/or 520 tagged with theelectroactive redox probe 525 to be covalently bound on a neutrallycharged PEG linker. When a host sample is introduced, theanti-immunoglobulin antibodies 510, 515, and/or 520 bind to either IgGantibodies 535, IgM antibodies 540, or IgA antibodies 545 in the sample.An anti-biofouling layer made of a hydrogel comprising charged PEGlayers 550 and lubricin (PRG4) 555 helps repel non-specific bindingproteins in the sample. When a positive potential is applied to theworking electrode 500, the proximity between the electroactive redox tag525 and the gold surface is modulated. The binding between theanti-antibodies 510, 515, and/or 520 and the IgG antibodies 535, IgMantibodies 540, or IgA antibodies 545 in the sample induces ahydrodynamic drag on the modulation, allowing the presence of IgGantibodies 535, IgM antibodies 540, or IgA antibodies 545 in the sampleto be confirmed using potentiometry measurements.

FIG. 6 shows a schematic illustration of a working electrode 600 of anexample electrochemical test strip device functionalized for anelectrochemical biosensor rapid antigen assay, again using SARS-CoV-2 asan example. The working electrode 600 comprises gold screen printed on asurface. Cu assisted click chemistry is used to covalently link theazido 605 modified monoclonal SARS-CoV-2 spike antigens 610 with anelectroactive tag 615 to the gold surface via a PEG-thiol linker with aterminal alkyne group 620. This allows preparation of electrode-tetheredantigens 610 tagged with the electroactive redox probe 615 to becovalently bound on a neutrally charged PEG linker. An anti-biofoulinglayer made of a hydrogel comprising charged PEG layers 625 and lubricin(PRG4) 630 helps repel non-specific binding analytes 635 in the sample.When a positive potential is applied to the working electrode 600, theproximity between the electroactive redox tag 615 and the gold surfaceis modulated. If antibodies 640 are present in the sample, such asproduced as a result of virus exposure or vaccination, these antibodies640 can bind to the tethered antigen 610, and the binding event canimpact the modulation rate induced by the application of potential tothe working electrode 600, leading to a detectable difference in currentoutput measured using potentiometry versus the non-binding configurationwhen the antibodies 610 are not present, indicating vaccine status orprior infection status.

FIG. 7 shows a schematic illustration of a working electrode 700 of anexample electrochemical test strip device functionalized for anelectrochemical biosensor rapid antigen assay, again using SARS-CoV-2 asan example. Cu assisted click chemistry is used to covalently link theazido 705 modified monoclonal SARS-CoV-2 nucleocapsid antigens 710 withan electroactive tag 715 to the gold surface via a PEG-thiol linker witha terminal alkyne group 720. This allows preparation ofelectrode-tethered antigens 710 tagged with the electroactive redoxprobe 715 to be covalently bound on a neutrally charged PEG linker. Ananti-biofouling layer made of a hydrogel comprising charged PEG layers725 and lubricin (PRG4) 730 helps repel non-specific binding analytes inthe sample. When a positive potential is applied to the workingelectrode 700, the proximity between the electroactive redox tag 715 andthe gold surface is modulated. With current SARS-CoV-2 vaccinesemploying spike antigens and not other SARS-CoV-2 antigens, adistinction between antibodies present due to infection or vaccinationcan be identified, as antibodies to the SARS-CoV-2 nucleocapscid proteinor SARS-CoV-2 envelope protein will likely only be in a test fluid ifthe test fluid is from an individual that has developed antibodies dueto infection by SARS-CoV-2. If antibodies 740 are present in the samplethat bind to the tethered antigen 710, such as produced as a result ofvirus exposure, the binding event can impact the modulation rate inducedby the application of potential to the working electrode 700, leading toa detectable difference in current output measured using potentiometryversus the non-binding configuration when the antibodies 710 are notpresent, indicating prior infection status.

In some cases, a working electrode of one set of electrodes in anelectrochemical test strip device can be functionalized with a vaccinespecific tethered antigen (e.g., as depicted in FIG. 6 ) and a workingelectrode of another set of electrodes in the electrochemical test stripdevice can be functionalized with an infection specific tethered antigen(e.g., as depicted in FIG. 7 ) to provide an electrochemical biosensorrapid antigen assay that distinguishes between immunity due tovaccination or immunity due to natural infection.

FIG. 8 shows a schematic illustration of an electrochemical biosensorbased multi-antigen assay using influenza and SARS-CoV-2 as examples.Bioprinter 800 is used for high speed printing of capture molecules(e.g., antibodies or antigens) on the surfaces of working electrodes inan electrochemical test strip device 805. The electrochemical test stripdevice strip 805 comprises two test chambers each with gold workingelectrodes 810 a and 810 b screen printed on the surface. Cu assistedclick chemistry is used to covalently link azido modified capturemolecules (e.g., monoclonal antibodies or antigens) with anelectroactive tag to the gold surface via a PEG-thiol linker with aterminal alkyne group. In some examples, the capture moleculesimmobilized in the first chamber are a cocktail of antibodies 815 aspecific to SARS-CoV-2 proteins and the monoclonal antibodiesimmobilized in the second chamber are antibodies 815 b specific toinfluenza proteins. This configuration allows for a differential signalto be measured based on the binding of the antibodies 815 a and 815 b totarget analytes (e.g., SARS-CoV-2 proteins or influenza proteins) and assuch can be used to distinguish between influenza and SARS-CoV-2 andprovide a rapid diagnosis of one infection condition versus the other.

FIG. 9 provides a schematic illustration of another electrochemicalbiosensor rapid antigen assay exemplified as a functionalizedelectrochemical test strip device 900. An electrochemical test stripdevice 900 includes two chambers—a test chamber 905 a and a referencechamber 905 b, each having a set of 3 electrodes and a reservoir forintroduction of test fluid. In the test chamber 905 a, active antibodies910 with electroactive tags 915 are immobilized on the surface ofworking electrode 920 a using click chemistry via a PEG linker, asdescribed above. In the reference chamber 905 b, null antibodies 925 areimmobilized on the surface of working electrode 920 b in a similarfashion. Using potentiostatic measurements, a difference in the currentoutput measured using the electrodes in the test chamber 905 a and thereference chamber 905 b when exposed to a test fluid can indicate thepresence or absence of a particular antigen.

FIG. 10 provides a schematic illustration of another electrochemicalbiosensor rapid antigen assay exemplified as a functionalizedelectrochemical test strip device 1000. Electrochemical test stripdevice 1000 includes two chambers—a test chamber 1005 a and a referencechamber 1005 b, each having a set of 3 electrodes and a reservoir forintroduction of test fluid. In the test chamber 1005 a, active receptorproteins 1010 with electroactive tags 1005 are immobilized on workingelectrode 1020 a using click chemistry via a PEG linker, as describedabove. In the reference chamber 1005 b, null receptor proteins 1025 withelectroactive tags are immobilized on the surface of working electrode1020 b in a similar fashion. Using potentiostatic measurements, adifference in the current output measured using the electrodes in thetest chamber 1005 a and the reference chamber 1005 b when exposed to atest fluid can indicate the presence or absence of a particular analyte1030 that binds to the active receptor protein 1010 or the null receptorprotein 1025.

FIG. 11 provides a schematic illustration of another electrochemicalbiosensor rapid antigen assay exemplified as a functionalizedelectrochemical test strip device 1100. Electrochemical test stripdevice 1100 includes two chambers—a test chamber 1105 a and a referencechamber 1105 b, each having a set of 3 electrodes and a reservoir forintroduction of test fluid. In the test chamber 1105 a, active receptoraptamers 1110 with electroactive tags 1105 are immobilized on workingelectrode 1120 a using click chemistry via a PEG linker, as describedabove. In the reference chamber 1105 b, null receptor aptamers 1125 withelectroactive tags are immobilized on the surface of working electrode1120 b in a similar fashion. Using potentiostatic measurements, adifference in the current output measured using the electrodes in thetest chamber 1105 a and the reference chamber 1105 b when exposed to atest fluid can indicate the presence or absence of a particular analytethat binds to the active receptor aptamer 1110 or the null receptoraptamer 1125.

FIG. 12 provides a schematic illustration of another electrochemicalbiosensor rapid antigen assay exemplified as a functionalizedelectrochemical test strip device 1200. Electrochemical test stripdevice 1200 includes two chambers—a test chamber 1205 a and a referencechamber 1205 b, each having a set of 3 electrodes and a reservoir forintroduction of test fluid. In the test chamber 1205 a, the antigensinclude SARS-CoV-2 spike antigens 1210, specific to antibodies produceddue to vaccination, coupled to an electroactive tag 1215, which isimmobilized on working electrode 1220 a using click chemistry via a PEGlinker, as described above. In the reference chamber 1205 b, theantigens include SARS-CoV-2 nucleocapsid antigens 1225, specific toantibodies produced due to virus exposure, coupled to an electroactivetag 1215, which is immobilized on the surface of working electrode 1220b in a similar fashion. Using potentiostatic measurements, a differencein the current output measured using the electrodes in the test chamber1205 a and the reference chamber 1205 b when exposed to a test fluid canindicate the presence or absence of a particular analyte that binds tothe spike antigen 1210 or the nucleocapsid antigen 1225. This assay canbe used to distinguish between subjects who have been vaccinated againstSARS-CoV-2 with a vaccine based on the spike antigen and those who wereinfected by SARS-CoV-2, which induces a humoral response against thespike antigen as well as the nucleocapsid antigen.

In an embodiment, an electrochemical test strip device described hereinobtains a fluid sample from an individual through physical contact. Forexample, an electrochemical test strip device may be placed in the mouthof an individual. In other examples, a collection device (e.g., adropper) is placed in the individual's mouth to collect saliva, which isthen applied to a fluid chamber of the electrochemical test stripdevice. In other examples, a collection device is used to obtain anotherfluid sample (e.g., blood, urine, etc.). The sample is placed into thefluid chamber where it contacts the functionalized working electrodes onthe electrochemical test strip device, which are subjected topotentiometry measurements by an interface device to obtain signalsindicative of a presence, absence, or concentration of target analytesin the fluid.

Interface Devices

The interface devices described herein are useful for obtaining andpresenting quantitative analog and digital signals generated fromelectrochemical measurements performed on the electrochemical teststrips described above. The interface devices can be used for evaluatingresults of testing analytes on electrochemical test strip devices andsending test results to an electronic device, such as a smartphone,tablet, or computer, for processing (e.g., to an application executingthereon) and communicating an output to the user. Information flow tothe electronic device can be achieved by a wired or wireless protocolsystem implemented on the interface device and the electronic device oran application executing thereon. The application or electronic devicemay be configured to provide qualitative (e.g., positive/negative) orquantitative results quickly, saving cost and time when tests need to beperformed at a high frequency. In some examples, an interface device foran electrochemical test strip device comprises a cable-based systemwhich can be used in combination with an electronic device.

Various embodiments of the interface device can accept a variety ofelectrochemical test strips. In some examples, electrochemical teststrips that can be accepted include electrochemical biosensor stripswith at two chambers with 3 electrodes each, as described herein, butother configurations with one or more than two chambers may be used. Ina specific example, an interface device comprises a smart cable havingdistal and proximal ends; the cable having a slot or port at the distalend, the slot or port capable of receiving an electrochemical test stripdevice; the cable having an electronic device adapter at the proximalend; a processor, the processor enclosed within the cable, the processorarranged to receive at least electronic signals and power from anelectronic device, and the processor capable of generating test resultsfrom the electrochemical test strip device. The electronic device may,in some examples, use Artificial intelligence based systems andalgorithms to compare raw analog data or digitized analog data toprevious test results to a similar demographic profile. In someexamples, the algorithm identifies a positive test result when a changein current from baseline is above a preset threshold from the previoustest results and a negative test result when the change in current frombaseline is below the preset threshold from the previous test results.The interface device may comprise a non-transitory computer readablememory storage device, such as enclosed within the cable. The memorystorage device may be capable of storing test results, firmware, orexecutable instructions, for example. The electronic device may delivera comparison report from the AI tools back to the user, such as with oneor more of a recommendation, a transaction log that providesnon-repudiation of the test results, metadata, a public/private keyencrypted hash based on an identifier for the electrochemical test stripdevice, which optionally contains details on the type of test,manufacturing history, timestamp, tester UserID, patient UserID.Optionally, an interface device includes an antenna (e.g., located inthe cable in the case of a cable-based interface device), such as anantenna that is capable of or configured to receive and transmit a testresult to another electronic device, such as via Bluetooth or otherwireless transmission protocol.

FIG. 13 provides an overview of an example interface device 1300 fordetecting analytes using an electrochemical test strip device. Interfacedevice 1300 may be constructed as a system on a chip (SoC). Examplecomponents of interface device 1300 may include a primary processor1305, a high speed standard bus 1310, a peripheral standard bus 1315, abus bridge 1320, a USB controller 1325, a power management unit 1330, awaveform generator 1335, a potentiostat 1340, an analog to digitalconverter (ADC) 1345, a digital to analog converter (DAC) 1350, internalnon-volatile memory 1355, a memory controller 1360 (e.g., forinterfacing with external memory), and a Bluetooth controller 1365.

The primary processor 1305 can run firmware or perform operations storedon non-voolatile memory 1355 or another non-transitory computer readablestorage medium. For example, the primary processor 1305 can execute ahierarchical state machine. For example, at any given time when there ispower, the primary processor 1305 can be booting, executing the statemachine, or in a sleep state. In examples, the primary processor 1305may be powered down and off when not in use. In some examples, power maybe provided, such as by connecting a USB cable, which powers up theprimary processor 1305 and causes it to execute a quick boot sequence.In one example, the primary processor 1305 obtains firmware frominternal memory 1355 and loads it into processor memory for execution.Once the boot sequence completes, the primary processor 1305 can beginrunning a state machine. In some examples, the state machine has only afew states at the top level: 1) Wait for Command, 2) Process Command, 3)Execute Test. The commands themselves can come from another deviceconnected to the interface device, such as a connected smartphone (e.g.,connected by USB or Bluetooth). Optionally, the commands may come fromone or more buttons located on or integrated with the interface device1300. When the primary processor 1305 receives a command, such as from asmartphone or a button, it goes into the Process Command state. In thisstate, the primary processor 1305 first decodes the command received,and then performs the appropriate operation. There may be a short listof commands available, including Provide Status, Execute BluetoothPairing, Run Test, Provide Test Data, and Clear Test Data.

If the command is to Run Test, the primary processor 1305 may proceedinto the Execute Test state. Execute Test is itself may be a statemachine, such as including the following states: 1) Pre-test Check, 2)Stimulus/Response, 3) Process Data, 4) Indicate Test Result, 5) SendTest Data. These states are described in further detail below.

The operation of the interface device 1300 may be coordinated by theprimary processor 1305, which can be a standard, low power, generalpurpose processor, such as an ARM processor or a similar RISCprocessors, ready to be compiled into the SoC design. The primaryprocessor 1305 controls the rest of the SoC system, operating a firmwareprogram which implements a hierarchical state machine, as describedbelow. The primary processor communicates 1305 with many of the othercomponents of the SoC using a standard high speed bus 1310, such as AMBAAHB. In embodiments, a standard bus, such as AMBA, is used as many othercomponents may be available and/or designed to work with AMBA.Advantageously, the bus is high speed to support real time datacollection. For example, during the Stimulus/Response portion of RunTest, the ADC 1345 will provide a steady stream of digital results data,which is transmitted via the high speed bus back 1310 to the primaryprocessor 1305. The primary processor 1305 also uses the high speed bus1310 to communicate with the USB controller 1325, sending data fortransmission over USB to the connected smartphone, or receiving commandsfrom the smartphone via USB. Similarly, the primary processor 1305 usesthe high speed bus to communicate with the Bluetooth controller 1365.The primary processor 1305 also uses the high speed bus for datatransactions with the internal memory 1355, and to interact with thememory controller 1360 for interactions with external memory. Finally,the primary processor 1305 uses the high speed bus 1310 to providecommands to the waveform generator 1335.

The peripheral standard bus 1315 may be implemented as a lower-speedstandard bus supporting a few peripherals that do not require thehigh-bandwidth communication provided by the high speed standard bus1310. This frees the high speed bus 1310 from needing to deal with thoseperipherals. A commonly-used standard bus for peripherals is the AMBAPeripherals Bus (APB). Examples of low speed peripherals in thisinterface device 1300 include LED drivers that control the usage ofindicator lights, and button controllers which provide de-bouncedsignals when buttons are pressed. The bus bridge 1320 manages theinterplay between the high speed bus 1310 and the peripherals bus 1315.An example of a bus bridge is the AMBA Bridge, used between AMBA highspeed bus and the APB.

The USB controller 1325 can provide functions, including enabling anexternal electronic device connected to the interface device to providepower through the USB connection. Also, the USB controller 1325 supportsdata communication between the connected electronic device and theinterface device. For this case, the USB controller 1325 translates thedata between the format and protocol used on the high speed standard bus1310 and the serial data and protocol defined by the USB standard.

The power management unit 1330 manages the power state of the interfacedevice 1330. To conserve power, the interface device 1330 can transitioninto a low power, sleep state after a predefined period of inactivity.The power management unit 1330 can also wake the interface device 1330back up from the low power state if it receives any interrupts from thebus bridge 1320, the USB controller 1325, or the Bluetooth controller1365, for example.

The waveform generator 1335 generates digital waveforms representingvoltage waveforms that are then sent through the DAC 1350 and to thepotentiostat 1340. An example of a digital waveform is the excitationwaveform used by square wave voltammetry. This is a digital rampwaveform with a specified start voltage, end voltage, and voltageincrement size and duration, with a superimposed square wave. To freethe primary processor 1305 from needing to manage all the details ofthese waveforms, the waveform generator 1335 can include an embeddedmicrocontroller. The primary processor 1305 sends commands to thewaveform generator 1335 to indicate the characteristics of the requiredwaveforms, and the waveform generator 1335 can generate the waveforms.In addition to generating the waveforms themselves, which then gothrough the DAC 1350 on the way to the potentiostat 1340, the waveformgenerator 1335 can also generate control signals to the ADC 1345 at theoutput of the potentiostat 1340. These control signals indicate when theADC 1345 is to take current samples from the potentiostat 1340. Thecurrent samples can be synchronized with the stimulus waveform, so thatthey are taken at the proper times. For example, for square wavevoltammetry two current samples at each voltage increment level areobtained: one taken near the end of the high phase of the square waveand one taken near the end of low phase of the square wave.

The potentiostat 1340 may be constructed as analog block includingfeedback amplifier circuits and precision resistors. The potentiostat1340 is configured to maintain a fixed voltage between the workingelectrode and reference electrode on the electrochemical test stripdevice while measuring current at the working electrode. Thepotentiostat 1340 connection to the reference electrode may beconfigured as a very high impedance connection, such that very littlecurrent flows from or to the reference electrode. A feedback amplifiermay be used to measure the actual potential between the workingelectrode and the reference electrode and compare it to the desiredpotential, as specified by the input from DAC 1350 at the output of thewaveform generator 1335. For example, the output can provide acorrection to keep the potential at the desired level.

The ADC 1345 converts analog current measurements from the potentiostat1340 to digital values which are then sent to the primary processor1305. The DAC 1350 converts digital signals coming from the waveformgenerator 1335 to analog voltage levels for the potentiostat 1340.

Pre-test Check: The Pre-Test Check can be configured as a short sequenceof steps to check that the interface device 1300 is set up properly toperform a test. One check is that a test strip is properly inserted intothe test strip connector and is properly filled with sample fluid. Theprimary processor 1305 can perform this step by commanding the waveformgenerator 1335 to provide a small DC voltage to apply across two of theelectrodes on the test strip and then getting the potentiostat 1340 andADC 1345 to measure the resulting current. The resulting analog currentmeasurement is converted to a DC value by the ADC 1345, and providedback to the primary processor as digital data. If there is no current,or a current much smaller than a threshold, it indicates either that thetest strip is not properly inserted, or that the sample chamber on thetest strip is not properly filled with fluid. If those conditions aretrue, the primary processor 1305 can generate a signal indicating an“invalid test” and abort the test, returning back to the Wait forCommand state. The signaling of “invalid test” optionally takesdifferent forms. In one example, the primary processor 1305 puts asignal onto the peripheral bus 1315 to specify that an “invalid testindicator” LED is to illuminate. In another example, the primaryprocessor sends an “invalid test” message via the USB controller 1325 orthe Bluetooth controller 1365 to a connected electronic device.

Stimulus/Response: In the Stimulus/Response state, the primary processor1305 provides commands to the waveform generator 1335 and receives datafrom the ADC 1345 after coming through the potentiostat 1340. Differentelectrochemical stimulus/response patterns are possible. For example,one stimulus/response approach is called square wave voltammetry. Inthat approach, the waveform generator 1335 is instructed to provide adigital voltage ramp, from a specified starting voltage to endingvoltage and with a specified step size. Superimposed on top of thatramp, the waveform generator 1335 provides a square wave whose periodcoincides with the duration of each voltage step of the ramp. Thepattern is fed into the potentiostat 1340, via the DAC 1350, and thepotentiostat 1340 applies this pattern as a set of voltage levelsbetween the working electrode and reference electrode of the connectedelectrochemical test strip device. The potentiostat 1340 can make twocurrent measurements at each voltage increment, one at the top of thesquare wave and one at the bottom. Those current measurements feedbackthrough the ADC 1345 to the primary processor 1305, where they arestored as digital results data in internal memory 1335.

In the Process Data state, the primary processor 1305 runs an analysisalgorithm on the collected test data to determine a test result. In someexamples, this analysis includes identifying peaks in the data, withinspecific ranges, assessing their amplitudes, comparing to other peaksand/or to thresholds, and filtering out noise. For examples where thereis a separate measurement chamber for reference, the data from thereference chamber are compared to that from the test chamber. If theanalysis determines that the results are above certain thresholds, thetest is considered positive. On the other hand, if the results arealigned with expectations and within thresholds for a negative, then thetest is considered negative. If neither of those is true, the test maybe considered invalid.

The Indicate Test Result state is used to communicate the test result.The communicate can occur in a variety of fashions. For example, theinterface device 1300 can provide a visual test result, such as byilluminating an LED light or generating a display on a display device(where included in interface device). In other examples, interfacedevice 1300 transmits the test result to a connected electronic deviceusing the USB controller 1325 or the Bluetooth controller 1365.

In the Send Test Data state, the primary processor 1305 sends thedigital form of the raw test data to the connected electronic device viaUSB or Bluetooth, for example. The Run Test state machine can optionallybe configured to automatically send the raw data after each test isperformed. In other configurations, the primary processor 1305 sends theraw data only when requested, using the Provide Test Data command.

FIG. 14 shows an illustration of an example handheld interface device1400. Electrochemical test strip devices may be connected to electrodecontacts 1405, accessible from a port at the edge of handheld interfacedevice 1400. With the top cover removed, the main circuit board 1410 isvisible, as is analog board 1415 and battery 1420. The main circuitboard can include a general purpose microcontroller unit with Bluetoothcapability, power management, memory, light emitting diode (LED)drivers, and switch/button inputs. The analog board 1415 can includepotentiostat capabilities (sensitive analog measurement components withprecise voltage control), waveform generator, ADCs and DACs, multiplexorcircuits, temperature sensors, or the like.

FIG. 15A and FIG. 15B show illustrations of a mobile electronic device1500, such as a smartphone, with an interface device 1505 connected to aport of the mobile electronic device 1500. Interface device 1505 isdepicted as an adapter-format test strip reader in FIG. 15A and as acable-based test strip reader in FIG. 15B (e.g., a Smart Cable). Anelectrochemical test strip device 1510 is shown inserted into a port ofthe interface device 1505, to allow for an electrochemical antigendetection assay to be performed, such as using potentiostaticmeasurements obtained using components integrated in interface device1505. Advantageously, mobile electronic device 1500 can include a cameraor other input device to allow for reading or input of a barcode oridentifier 1515 that may be present on electrochemical test strip device1510. Mobile electronic device 1500 can include a display that canprovide a test result 1520 or provide another barcode 1525 that can beused to identify a particular sample and/or the test result.

FIG. 16 provides an overview of a wide-scale pandemic Real-Time TestingSystem (RTTS) 1600, which can be used with electrochemical biosensorrapid antigen assays and electrochemical test strip devices describedherein and used for real-time screening for entry into venues, events,transportation systems, or the like. The RTTS 1600 includes aPoint-of-Entry Testing Device 1605, which can be used to obtain testresults or screening results for an infectious disease (e.g., infectionby SARS-CoV-2) or vaccination or prior infection by an infectiousdisease. RTTS 1600 can employ an electrochemical test strip device, asdescribed herein, for device 1605 used for screening. In the case of apositive result 1610 of screening (e.g., indicating infection bySARS-CoV-2), nonaccess to the venue, event, or transportation system istriggered by RTTS 1600. In the case of a negative result 1615 ofscreening (e.g., indicating non-infection by SARS-CoV-2), access to thevenue, event, or transportation system is triggered by RTTS 1600. Accessto the venue, event, or transportation system can, in some cases can beelectronically permitted or restricted by an access device (e.g., alocking door, turnstyle, etc.) or by displaying an audible or visibleindicator for use by an usher to permit or deny access to the venue,event, or transportation system.

In some examples, an authenticating application (App) 1620 is executedon a smartphone or computer, which may be connected to thePoint-of-Entry Testing Device 1605 by wired connector or wireless means(such as, but not limited to, Bluetooth and WiFi). The authenticatingApp 1620 may be configured to use government identification (e.g.,driver license, passport, identity card, etc.), biometric scan (e.g.,fingerprint, voice print, facial recognition, etc.), or other way forverifying identity of an individual testee or association with a testresult for an electrochemical biosensor rapid antigen assay. Acloud-based server App 1625, stores information from a database,including, but not limited to, Testee ID, Testee results, Testee Site,time and date stamp and GPS data; the cloud-based Server App is furthercapable of interacting with Third-Party apps 1630, such as for thepurpose of providing a Digital Results Badge of the results (structuredin typical formats including but not limited to HTML, CSV, text, andExcel) for authenticating Testee at sites other than the originalPoint-of-Entry device. The Artificial Intelligence analysis app 1635,can integrate data from the cloud-based Server App 1625 and apps forGeographical Information Services (GIS), and population data, GPS data,and location systems. The machine learning system 1640 or ArtificialIntelligence Analysis App 1635 and associated algorithms can interpretdata in real-time to provide confidence intervals and learn from pasttrends to make predictions on future outcomes, which may be referred toas a a Real-Time Processing System (RTPS). External Queries App 1645, isthe human (for example but not limited to Web page or Smartphone App)and computer interface (for example but not limited to an ApplicationProgramming Interface aka API) allowing the Real-Time Testing System1600 to be interrogated and data downloaded.

Electrochemical Test Data and Results

FIG. 17A provides voltammogram data obtained using an electrochemicaltest strip device functionalized with SARS-CoV-2 S1 monoclonalantibodies as capture molecules when exposed to test fluids containingno (e.g., only buffer) and varying concentrations of recombinantSARS-CoV-2 S1+S2 ECD spike antigens. The data shows a change in measuredcurrent outputs and indicates that, as the concentration of the antigensincreases, the current output decreases.

FIG. 17B provides a dose response curve with standard deviations whenthe immobilized S1 monoclonal antibody was tested against recombinantS1+S2 ECD spike antigens. Square Wave Voltammetry confirms change incurrent signal with a limit of detection (LOD) as low as 1 femtogram permilliliter of the antigen and a dynamic range of 1 femtogram/ml to 10nanogram/ml.

FIG. 18 provides voltammogram data obtained using an electrochemicaltest strip device functionalized with SARS-CoV-2 antibodies as capturemolecules tested against SARS-CoV-2 spike antigens vs influenzaneuraminidase antigens as test analytes. The results show specificity ofthe test to SARS-CoV-2 spike antigens vs influenza neuraminidaseantigens. In the case of the influenza antigens, there was no observedchange in signal as compared to a buffer solution, while the SARS-CoV-2antigens caused a decrease in signal, exhibiting specificity of thefunctionalized electrochemical test strip for testing for SARS-CoV-2.

FIG. 19A provides voltammogram data obtained using an electrochemicaltest strip device functionalized with SARS-CoV-2 S1 monoclonalantibodies as capture molecules tested against heat inactivated viruses(2019-nCoV/USA/WA1/2020) in buffer, showing variations in currentoutputs for different concentrations of the heat inactivated viruses. Asthe concentration of the heat inactivated virus increases, the currentoutput decreases. Initial testing using recombinant S1/S2 antigenfragments in buffer indicated a quantitative testing window withsaturation but no hook effect, which was confirmed using heatinactivated virus.

FIG. 19B provides a dose response curve with standard deviations whenthe immobilized S1 monoclonal antibody was tested against wild-type heatinactivated viruses in buffer. The results confirm the change in currentsignal, with a limit of detection (LOD) as low as 0.1 TCID₅₀/ml.

FIG. 20 provides data showing the current output obtained using anelectrochemical test strip functionalized with an active capturemolecule and tested using saliva as a test fluid with buffer solution asa reference. Compared to buffer solution, saliva reduced the detectedcurrent output as though high concentration of a binding analyte werepresent, and these results were attributed to biofouling by nonspecificanalytes (e.g., mucins and amylases) in the saliva sample. Differentstrategies were evaluated to for preventing electrode biofouling,including coating the working electrode surface with additionalanti-fouling agents or using other anti-fouling techniques. Bovine serumalbumin was reported in literature as an anti-fouling agent but did notshow effective results in saliva. Dilution of the saliva did not producegood current output and the effect of electrode fouling was seen in lessthan 2 minutes. Starch filtration removed the amylases but had no effecton mucins. Using PEG layers provided a current output which wassignificantly smaller when compared to baseline signal generated in thepresence of a buffer. PRF1 consisting of recombinant PRG4 (rPRG4) andneutral PEG layers provided an increase in current output in thepresence of saliva but was not successful in regaining the baselinecurrent output. PRF2 consisting of recombinant proteoglycan 4 (PRG4) andnegatively charged PEG layers provided a comparable current output inthe presence of both buffer and saliva.

FIG. 21 provides data showing the current output with increasingconcentrations of heat inactivated virus in pooled saliva when usingelectrochemical test strips modified with PRF2. From the data, it can bedetermined that the LOD is about 0.1 TCID₅₀/ml and the highestquantifiable concentration at saturation is 100 TCID₅₀/ml, therebyproducing a 3-log quantification range. To compare the LOD from thistechnique with that of standard rapid antigen tests, BD Veritor PlusAnalyzer and BinaxNOW COVID-19 Ag Card have an estimated limit ofdetection at 140 TCID₅₀/ml. The FDA cleared BioFire Respiratory Panel2.1 PCR test has a SARS-CoV-2 LOD for heat-inactivated virus (WA1/2020)of 6.9×10⁻² TCID₅₀/ml.

FIG. 22 provides data from a blind study that was performed usingcontrived human saliva samples. The samples were spiked with varying,unknown concentrations of positive and negative controls from Sofia SARSAntigen FIA Control Swabs and tested against an electrochemical teststrip device functionalized with SARS-CoV-2 antibodies as capturemolecules as a test for SARS-CoV-2. The negative control swab consistsof heat-inactivated Group C Streptococcus while the positive controlswab consisted of non-infectious recombinant SARS-CoV-2 antigen. Thepositive contrived samples were further spiked with various unknownconcentrations of heat inactivated SARS-CoV-2 viruses. Both controlswabs were verified using the BD Veritor kit for confirmation aspositive or negative. The test for SARS-CoV-2 shows a clear distinctionbetween positive and negative samples. The results displayed show thedifference in signal response between positive and negative samplesdemonstrating the functionality of the electrochemical test stripdevices, where saliva was placed into the fluid chamber. An interfacedevice was used to measure the signal from the electrochemical teststrip devices and the results were transferred via Bluetooth to a testapplication for processing. Overall, from sample collection to results,these results were obtained in about 1.5 minutes: 45 sec for collection,30 sec equilibration time and 15 sec to obtain results.

FIG. 23A provides voltammogram data and FIG. 23B provides current outputdata obtained using an electrochemical test strip device functionalizedwith SARS-CoV-2 S1 monoclonal antibodies as capture molecules testedagainst SARS-CoV-2 variant antigens with different mutations andcomparison with a null sample (no antigens) as a reference. Thewild-type SARS-CoV-2 showed the strongest response in these results,with different variants showing strong signals compared to thereference, indicating the utility of the electrochemical test stripdevice for identifying infection by SARS-CoV-2 variants.

FIG. 24 provides voltammogram data obtained using an electrochemicaltest strip device functionalized with methylene blue tagged ACE2proteins as capture molecules. Different passivation layers were testedto determine the maximum current output based on the presence or absenceof charge on the PEG layers. It was determined that the negativelycharged PEG layer is useful in repelling similar charged molecules inthe sample, showing a matrix effect in buffer and producing backgroundnoise. On the contrary, neutral PEG layers were observed to have a lowerbackground with higher current output. While both types of PEG layersfacilitated good current output, neutral PEG layer had the highercurrent output and lower background. For other studies, charged PEGlayer can be used as the passivation layer.

FIG. 25 provides voltammogram data from a validation test obtained usingan electrochemical test strip device functionalized with methylene bluetagged ACE2 proteins as capture molecules. A solution of 10 ng/ml ofRabbit ACE2 monoclonal antibody in PBS buffer was used to validate thebinding affinity as compared to a phosphate-buffered saline (PBS) bufferwith no antibody present. A decrease in the current output associatedwith the binding event is indicative that the ACE2 protein immobilizedon the working electrode of the electrochemical test strip remainsactive.

FIG. 26 provides voltammogram data from another validation testperformed to determine if the receptor binding domain (RBD) antigen isactive. Two different RBD antigens were used for thisexperiment—Recombinant human coronavirus SARS-CoV-2 Spike GlycoproteinRBD (Active) (ab273065) and Recombinant Human coronavirus SARS-CoV-2Spike Glycoprotein RBD (His tag) (ab275986). Out of the two antigens,only Recombinant human coronavirus SARS-CoV-2 Spike Glycoprotein RBDshowed binding while the other antigen did not show any activity. Usingthe active RBD antigen, a dose response was generated at concentrationsof 10 fg/ml, 10 pg/ml, and 10 ng/ml using an electrochemical test stripfunctionalized with immobilized ACE2 protein as a capture molecule.There was significant decrease in current output when the RBD antigenswere added to the strip, indicating binding and active RBD antigens.

FIG. 27A provides voltammogram data and FIG. 27B provides dose responsecurve data where ACE2 protein was immobilized on the working electrodeof an electrochemical test strip that was evaluated for binding with RBDantigens at different concentrations. As the concentration of the S1-RBDantigens was increased, the current output decreased. A significantdifference was observed in current output between the buffer withoutantigens and 0.1 fg/ml, which indicates that the lower limit ofdetection is 0.1 fg/ml and the highest detectable concentration was 10ng/ml of the antigens. Using the concentration beyond 10 ng/ml does notcause any further decrease in the peak height.

FIG. 28A provides voltammogram data and FIG. 28B provides current outputdata obtained using an electrochemical test strip device functionalizedwith ACE2 protein as capture molecules tested against SARS-CoV-2 variantantigens with different mutations and comparison with a null sample (noantigens) as a reference. Variants with N501Y mutation showed higherbinding to the ACE2 protein while the K417T and E484K mutation exhibiteddecreased binding affinity which is reflected in the current outputsthat indicate binding.

FIG. 29 provides comparative data showing differences in bindingaffinity based on using different capture molecules—ACE2 protein, spikeRBD specific monoclonal antibody, and spike S1-subunit monoclonalantibody. While ACE2 protein, spike RBD monoclonal antibody and spikeS1-subunit monoclonal antibody have similar binding affinities at lowconcentrations, spike S1-subunit monoclonal antibody has better bindingaffinity at higher concentrations of the recombinant S1+S2 extracellulardomain antigens.

FIG. 30 provides comparative data showing differences in bindingaffinity based on using different capture molecules—ACE2 protein, spikeRBD specific monoclonal antibody, and spike S1-subunit monoclonalantibody. Results show that all three capture agents—ACE2 protein, spikeRBD monoclonal antibody and spike S1-subunit monoclonal antibody havesimilar binding affinities to spike RBD antigens.

The invention may be further understood by the following non-limitingexamples.

Example 1

This Example describes an Electrochemical Biosensor Antigen Assay fortroponin I utilizing instant-readout potentiostats using a directantigen binding assay format. Gold surface is functionalized using athiolated PEG linker that binds the azido modified monoclonalanti-troponin antibody labeled with an electroactive tag and thenincubated at room temperature for 1 hour. For the assay, whole blood isobtained by venipuncture from suspects suspected of having an acute MIand tested with the biosensor. Whole blood is aliquoted before clottinginto the sample collection tube provided (300 mL), and the cap is placedon the tube assembly. Approximately 6 drops are applied to the testchamber. 0.4 ng/ml cardiac troponin solution is added to the referencechamber. Results of the test are read immediately by quantitationutilizing biosensor instant-readout square wave voltammetry. Theresponse is immediately reported as amount of troponin antigen higher orlower than threshold concentration in the sample.

Example 2

This Example describes an Electrochemical Biosensor Antigen Assay fortroponin I utilizing instant-readout potentiostats using a directantigen binding assay format. Gold surface is functionalized usingtroponin I binding aptamers. For the assay, whole blood is obtained byvenipuncture from suspects suspected of having an acute MI and testedwith the biosensor. Whole blood is aliquoted before clotting into thesample collection tube provided (300 mL), and the cap is placed on thetube assembly. Approximately 6 drops are applied to the sensor port.Results of the test are read immediately by quantitation or as positiveor negative utilizing biosensor instant-readout square wave voltammetry.The response is immediately reported as amount of troponin antigen inthe sample.

Example 3

This Example describes an Electrochemical Biosensor for Multiple AntigenAssay for the cardiac marker's troponin I, CKMB, and BNP by bothqualitative and quantitative biosensor means. Gold surface isfunctionalized using a thiolated PEG linker that binds a cocktail ofazido modified antibodies for troponin I, CKMB, and BNP in an equimolarratio. Each antibody is labeled with a different electroactive tagnamely E1, E2 and E3. For the assay, whole blood is obtained byvenipuncture from suspects suspected of having an acute MI and testedwith the biosensor. Whole blood is aliquoted before clotting into thesample collection tube provided (300 mL), and the cap is placed on thetube assembly. Approximately 6 drops are applied to the test chamber. Areference solution comprising 0.4 ng/ml of cardiac troponin I, 1.6 ng/mlof CKMB and 100 pg/ml of BNP is added to the reference chamber. Resultsof the test are read immediately by quantitation or as positive ornegative utilizing biosensor instant-readout square wave voltammetry. heresponse is immediately reported as amount of troponin I, CKMB and BNPhigher or lower than threshold concentration in the sample.

Example 4

This Example describes an Electrochemical Biosensor for Multiple AntigenAssay for the cardiac marker's troponin I, CKMB, and BNP by bothqualitative and quantitative biosensor means. Gold surface isfunctionalized using aptamers for troponin I, CKMB, and BNP in anequimolar ratio. Each aptamer is labeled with a different electroactivetag namely E1, E2 and E3. For the assay, whole blood is obtained byvenipuncture from suspects suspected of having an acute MI and testedwith the biosensor. Whole blood is aliquoted before clotting into thesample collection tube provided (300 mL), and the cap is placed on thetube assembly. Approximately 6 drops are applied to the sensor port.Results of the test are read immediately by quantitation or as positiveor negative utilizing biosensor instant-readout square wave voltammetry.The response is immediately reported as amount of troponin I, CKMB andBNP in the sample.

Example 5

This Example describes an Electrochemical Biosensor for SARS-CoV-2formed from a gold-bound DNA aptamer that binds to antigen, such asSARS-CoV-2 spike protein S1 and/or S2 or nucleocapsid protein, havingone end (for one example, the 5′ end) with a thiol (—SH) moiety thatbinds to the gold surface and the opposite end (in this case the 3′ end)having an electroactive tag such as methylene blue or ferrocene that isfree to move. Binding of the Sars-CoV-2 antigen results in aconformational change in the aptamer moving the electroactive tagrelative to the charged gold surface resulting in an electrical signal.The change in signal, or lack thereof, is indicative of the presence orabsence of SARS-CoV-2 particles and detection of the disease.

REFERENCES

-   U.S. Pat. Nos. 7,316,766, 8,309,345, 8,737,971, 9,257,038,    9,366,645, 9,445,749, 9,686,395, 9,787,815, 9,808,798, 10,572,627,    and 11,112,412.-   U.S. Patent Application Publication Nos. 2013/0059293, 2014/0014536,    2014/0294675, 2016/0041146, 2016/0363550, 2018/0188244,    2019/0170738, 2020/0333286, 2021/0003528, 2021/0093248, and    2021/0055259.-   PCT International Application Publication Nos. WO 2018/223090, WO    2019/005473, WO 2020/097138, and WO 2020/186118.-   Chinese Patent Documents CN111024954A, CN111537746A.-   European Patent Document EP3855186A2-   Indian Patent Document IN202021016563A.-   Yang, J. et al., Appl. Phys. Lett. 111, 2021049, 2017.-   Yang, J. et al., Appl. Phys. Lett. 113, 032101, 2018.-   Carey, P. et al., J. Electrochemical Soc. 166, (8) B708-B712, 2019.-   Chiang et al., Development and validation of a quantitative,    non-invasive, highly sensitive and specific, electrochemical assay    for anti-SARS-CoV-2 IgG antibodies in saliva′-   Bell J A, May F E, Stewart R B: Clinical research in the elderly:    Ethical and methodological considerations. Drug Intelligence and    Clinical Pharmacy, 21: 1002-1007, 1987.-   Cosgrove R: Understanding drug abuse in the elderly. Midwife, Health    Visitor & Community Nursing 24(6):222-223, 1988.-   Koyoma, T. et al., Bulletin of the WHO, 98(7), 2020

STATEMENTS REGARDING INCORPORATION BY REFERENCE AND VARIATIONS

All references throughout this application, for example patent documentsincluding issued or granted patents or equivalents; patent applicationpublications; and non-patent literature documents or other sourcematerial; are hereby incorporated by reference herein in theirentireties, as though individually incorporated by reference, to theextent each reference is at least partially not inconsistent with thedisclosure in this application (for example, a reference that ispartially inconsistent is incorporated by reference except for thepartially inconsistent portion of the reference).

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains. References cited herein are incorporated byreference herein in their entirety to indicate the state of the art, insome cases as of their filing date, and it is intended that thisinformation can be employed herein, if needed, to exclude (for example,to disclaim) specific embodiments that are in the prior art. Forexample, when a compound is claimed, it should be understood thatcompounds known in the prior art, including certain compounds disclosedin the references disclosed herein (particularly in referenced patentdocuments), are not intended to be included in the claim.

When a group of substituents is disclosed herein, it is understood thatall individual members of those groups and all subgroups and classesthat can be formed using the substituents are disclosed separately. Whena Markush group or other grouping is used herein, all individual membersof the group and all combinations and subcombinations possible of thegroup are intended to be individually included in the disclosure. Asused herein, “and/or” means that one, all, or any combination of itemsin a list separated by “and/or” are included in the list; for example,“1, 2, and/or 3” is equivalent to “‘1’ or ‘2’ or ‘3’ or ‘1 and 2’ or ‘1and 3’ or ‘2 and 3’ or ‘1, 2, and 3’”.

Every formulation or combination of components described or exemplifiedcan be used to practice the invention, unless otherwise stated. Specificnames of materials are intended to be exemplary, as it is known that oneof ordinary skill in the art can name the same material differently. Oneof ordinary skill in the art will appreciate that methods, deviceelements, starting materials, and synthetic methods other than thosespecifically exemplified can be employed in the practice of theinvention without resort to undue experimentation. All art-knownfunctional equivalents, of any such methods, device elements, startingmaterials, and synthetic methods are intended to be included in thisinvention. Whenever a range is given in the specification, for example,a temperature range, a time range, or a composition range, allintermediate ranges and subranges, as well as all individual valuesincluded in the ranges given are intended to be included in thedisclosure.

As used herein, “comprising” is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps. As usedherein, “consisting of” excludes any element, step, or ingredient notspecified in the claim element. As used herein, “consisting essentiallyof” does not exclude materials or steps that do not materially affectthe basic and novel characteristics of the claim. Any recitation hereinof the term “comprising”, particularly in a description of components ofa composition or in a description of elements of a device, is understoodto encompass those compositions and methods consisting essentially ofand consisting of the recited components or elements. The inventionillustratively described herein suitably may be practiced in the absenceof any element or elements, limitation or limitations which is notspecifically disclosed herein.

The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention in the useof such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, but it is recognizedthat various modifications are possible within the scope of theinvention claimed. Thus, it should be understood that although thepresent invention has been specifically disclosed by preferredembodiments and optional features, modification and variation of theconcepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

What is claimed is:
 1. An electrochemical test strip device comprising:a first set of electrodes including first working electrodefunctionalized with active capture molecules, wherein the active capturemolecules are directly labeled with a plurality of electroactive redoxtags, and wherein the active capture molecules are configured to bind atarget analyte; a second set of electrodes including a second workingelectrode functionalized with null capture molecules, wherein the nullcapture molecules are directly labeled with a plurality of electroactiveredox tags, and wherein the null capture molecules are configured not tobind the target analyte; a fluid chamber in fluid communication with thefirst set of electrodes and the second set of electrodes, the fluidchamber for receiving a test fluid including one or more test analytesand establishing contact between the test fluid and the first set ofelectrodes and between the test fluid and the second set of electrodes,wherein the first set of electrodes and the second set of electrodes areconfigured to provide a differential measurement for identifying thetarget analyte from the one or more test analytes; wherein the firstworking electrode includes a first heterogeneousself-assembled-monolayer (SAM) including a first linker component and acharged passivation component, wherein the charged passivation componentis bound to a surface of the first working electrode, the first linkercomponent terminally binding the active capture molecules on one end andalso binding a surface of the first working electrode, wherein the firstlinker component and active capture molecule comprises a click chemistryadduct or bioconjugate; wherein the second working electrode includes asecond heterogeneous SAM including a second linker component and thecharged passivation component, wherein the charged passivation componentis bound to a surface of the second working electrode, the second linkercomponent terminally binding the null capture molecules on one end andalso binding a surface of the second working electrode, wherein thesecond linker component and null capture molecule comprises a clickchemistry adduct or bioconjugate; and wherein the first heterogeneousSAM and the second heterogeneous SAM each further comprises ananti-fouling formulation comprising a plurality of different chargedglycosaminoglycans bound to surfaces of the first working electrode andthe second working electrode and interspersed between the first linkercomponent and the charged passivation component and between the secondlinker component and the charged passivation component.
 2. Theelectrochemical test strip device of claim 1, wherein theelectrochemical test strip device comprises an antigen assay for a virusor a component thereof, a viral biomarker, or an inflammatory biomarker,wherein the target analyte is the virus or the component thereof, theviral biomarker, or the inflammatory biomarker, wherein the activecapture molecules include a first antibody that binds the virus or thecomponent thereof, the viral biomarker, or the inflammatory biomarker,or a receptor protein that binds the virus or the component thereof, theviral biomarker, the inflammatory biomarker, and wherein the nullcapture molecules include a second antibody that does not bind the virusor the component thereof, the viral biomarker or the inflammatorybiomarker.
 3. The electrochemical test strip device of claim 2, whereinthe virus is a coronavirus, wherein the viral biomarker is a coronavirusspike protein or a coronavirus nucleocapsid protein, or wherein theinflammatory biomarker is a coronavirus-induced inflammatory biomarkeror host protein.
 4. The electrochemical test strip device of claim 3,wherein the virus is SARS-CoV-2 or a variant of SARS-CoV-2, wherein theviral biomarker comprises a spike protein or a nucleocapsid protein ofSARS-CoV-2 or a variant of SARS-CoV-2, or wherein the inflammatorybiomarker comprises a cytokine, a SARS-CoV-2-mediated inflammatorybiomarker, or a SARS-CoV-2 variant-mediated inflammatory biomarker. 5.The electrochemical test strip device of claim 2, wherein the firstantibody comprises an anti-SARS-CoV-2 antibody, an anti-SARS-CoV-2variant antibody, or an anti-cytokine antibody, or wherein the receptorprotein comprises an angiotensin-converting enzyme 2 (ACE-2) or portionthereof.
 6. The electrochemical test strip device of claim 1, whereinthe electrochemical test strip device comprises an assay for detectionof viral antibodies or antiviral vaccine-induced antibodies, wherein thetarget analyte is viral infection-developed antibody or an antiviralvaccine-developed antibody, and wherein the active capture moleculesinclude a viral structural protein or portion thereof.
 7. Theelectrochemical test strip device of claim 6, wherein the viralinfection-developed antibody comprises a coronavirus infection-developedantibody, wherein the antiviral vaccine-developed antibody comprises ananti-coronavirus vaccine-developed antibody, or wherein the viralstructural protein comprises a coronavirus structural protein.
 8. Theelectrochemical test strip device of claim 7, wherein the viralinfection-developed antibody comprises a SARS-CoV-2 infection-developedantibody or a SARS-CoV-2 variant infection-developed antibody, whereinthe antiviral vaccine-developed antibody comprises an anti-SARS-CoV-2vaccine-developed antibody or anti-SARS-CoV-2 variant vaccine-developedantibody, or wherein the viral structural protein comprises a SARS-CoV-2or SARS-CoV-2 variant spike protein, a SARS-CoV-2 or SARS-CoV-2 variantnucleocapsid protein, or a SARS-CoV-2 or SARS-CoV-2 variant envelopeprotein.
 9. The electrochemical test strip device of claim 6, whereinthe viral structural protein comprises a SARS-CoV-2 spike protein, aSARS-CoV-2 nucleocapsid protein, or a SARS-CoV-2 envelope protein, andwherein the null capture molecules include a SARS-CoV-2 variant spikeprotein or portion thereof, a SARS-CoV-2 variant nucleocapsid protein orportion thereof, or a SARS-CoV-2 variant envelope protein or portionthereof.
 10. The electrochemical test strip device of claim 1, whereinthe active capture molecules include a first antibody, first receptorprotein, or a first aptamer that binds the target analyte, and whereinthe null capture molecules include a second antibody, second receptorprotein, or a second aptamer that does not bind the target analyte. 11.The electrochemical test strip device of claim 1, wherein the pluralityof different charged glycosaminoglycans prevent electrode biofouling byrepelling non-specific proteins in the test fluid.
 12. An interfacedevice comprising: a set of electrode contacts for coupling to one ormore electrode contacts of an electrochemical test strip device, whereinthe electrochemical test strip device is the electrochemical test stripdevice of claim 1; one or more potentiostats in electrical communicationwith the set of electrode contacts; and a waveform generator inelectrical or control communication with the one or more potentiostatsfor generating a potential waveform.
 13. The interface device of claim12, further comprising: a processor in data or control communicationwith the one or more potentiostats and the waveform generator, whereinthe processor is programmed with instructions or in data communicationwith a non-transitory computer-readable storage medium storing processorexecutable instructions, wherein the instructions, when executed by theprocessor, cause the processor to perform operations including:obtaining voltammograms using the one or more potentiostats, thewaveform generator, the first set of electrodes, and the second set ofelectrodes.
 14. The interface device of claim 13, wherein thevoltammograms include a test voltammogram obtained using the set ofelectrodes or a first portion thereof and a reference voltammogramobtained using the set of electrodes or a second portion thereof. 15.The interface device of claim 13, wherein the operations further includereceiving input corresponding to a directive to initiate obtaining thevoltammograms.
 16. The interface device of claim 15, wherein theoperations further include outputting a test result corresponding to apresence, absence, or quantitative measure of the target analyte in thetest fluid based on the voltammograms within 1-5 minutes after receivingthe input.
 17. The interface device of claim 13, wherein the operationsfurther include outputting a test result corresponding to a presence,absence, or quantitative measure of the target analyte in the test fluidbased on the voltammograms.
 18. The interface device of claim 13,wherein the test result has a sensitivity or detection limit for thetarget analyte of from 0.1 TCID₅₀/ml to about 10 TCID₅₀/ml or asensitivity or detection limit for the target analyte of from about 1fg/ml to about 1 pg/ml.
 19. A kit comprising: the electrochemical teststrip device of claim 1; and instructions for applying a test fluid tothe test chamber and for coupling the electrochemical test strip deviceto an interface device to obtain a test result indicating a presence,absence, or quantitative measure of the target analyte in the testfluid.
 20. The kit of claim 19, further comprising: the interface devicefor coupling to the electrochemical test strip device, wherein theinterface device comprises: a third set of electrode contacts forcoupling to the first set of electrode contacts and the second set ofelectrode contacts of the electrochemical test strip device; one or morepotentiostats in electrical communication with the third set ofelectrode contacts; and a waveform generator in electrical or controlcommunication with the one or more potentiostats for generating apotential waveform.